Activatable antibodies that bind epidermal growth factor receptor and methods of use thereof

ABSTRACT

The invention relates generally to activatable antibodies that include a masking moiety (MM), a cleavable moiety (CM), and an antibody (AB) that specifically binds to epidermal growth factor receptor (EGFR), and to methods of making and using these anti-EGFR activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/875,947, filed Jan. 19, 2018, which is a divisional of U.S. patentapplication Ser. No. 15/381,972, filed Dec. 16, 2016 and now U.S. Pat.No. 9,889,211, which is a divisional of U.S. patent application Ser. No.14/815,111, filed Jul. 31, 2015 and now issued as U.S. Pat. No.9,545,442, which is a divisional of U.S. patent application Ser. No.13/872,052, filed Apr. 26, 2013 and now issued as U.S. Pat. No.9,120,853, which claims the benefit of U.S. Provisional Application No.61/639,796, filed Apr. 27, 2012; U.S. Provisional Application No.61/662,204, filed Jun. 20, 2012; U.S. Provisional Application No.61/749,220, filed Jan. 4, 2013; U.S. Provisional Application No.61/749,529, filed Jan. 7, 2013; and U.S. Provisional Application No.61/763,237, filed Feb. 11, 2013; each of which is incorporated herein byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “CYTM017D04US SeqList.txt”, whichwas created on Jun. 3, 2020 and is 60.0 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to activatable antibodies thatspecifically bind to epidermal growth factor receptor (EGFR), conjugatedactivatable antibodies that specifically bind to EGFR, and methods ofmaking and using these anti-EGFR activatable antibodies and/orconjugated anti-EGFR activatable antibodies in a variety of therapeutic,diagnostic and prophylactic indications.

BACKGROUND OF THE INVENTION

Antibody-based therapies have proven effective treatments for somediseases but in some cases, toxicities due to broad target expressionhave limited their therapeutic effectiveness. In addition,antibody-based therapeutics have exhibited other limitations such asrapid clearance from the circulation following administration.

In the realm of small molecule therapeutics, strategies have beendeveloped to provide prodrugs of an active chemical entity. Suchprodrugs are administered in a relatively inactive (or significantlyless active) form. Once administered, the prodrug is metabolized in vivointo the active compound. Such prodrug strategies can provide forincreased selectivity of the drug for its intended target and for areduction of adverse effects.

Accordingly, there is a continued need in the field of antibody-basedtherapeutics for antibodies that mimic the desirable characteristics ofthe small molecule prodrug.

SUMMARY OF THE INVENTION

The invention provides activatable antibodies that include an antibodyor antigen-binding fragment thereof that specifically binds epidermalgrowth factor receptor (EGFR) coupled to a masking moiety (MM), suchthat coupling of the MM reduces the ability of the antibody orantigen-binding fragment thereof to bind EGFR. In a preferredembodiment, the MM is coupled via a sequence that includes a substratefor a protease, for example, a protease that is co-localized with EGFRat a treatment site in a subject. The activatable anti-EGFR antibodiesprovided herein are stable in circulation, activated at intended sitesof therapy and/or diagnosis but not in normal, i.e., healthy tissue,and, when activated, exhibit binding to EGFR that is at least comparableto the corresponding, unmodified antibody.

The invention provides conjugated activatable antibodies that include anantibody or antigen-binding fragment thereof that specifically bindsepidermal growth factor receptor (EGFR) coupled to a masking moiety(MM), such that coupling of the MM reduces the ability of the antibodyor antigen-binding fragment thereof to bind EGFR, and wherein theactivatable antibody is conjugated to one or more additional agents. Ina preferred embodiment of the conjugated activatable antibodies, the MMis coupled via a sequence that includes a substrate for a protease, forexample, a protease that is co-localized with EGFR at a treatment sitein a subject. The conjugated, activatable anti-EGFR antibodies providedherein are stable in circulation, activated at intended sites of therapyand/or diagnosis but not in normal, i.e., healthy tissue, and, whenactivated, exhibit binding to EGFR that is at least comparable to thecorresponding, unmodified antibody.

The invention provides methods of treating, preventing and/or delayingthe onset or progression of, or alleviating a symptom of an indication,e.g., disease or disorder, associated with aberrant expression and/oractivity of EGFR, e.g., an EGFR-related disorder, in a subject using atherapeutic molecule, e.g., activatable antibodies that in an activatedstate bind EGFR and/or conjugated activatable antibodies that in anactivated state bind EGFR, particularly activatable antibodies and/orconjugated activatable antibodies that when activated bind andneutralize or otherwise inhibit at least one biological activity of EGFRand/or EGFR-mediated signaling.

The activatable antibodies described herein in an activated state bindEGFR and include (i) an antibody or an antigen binding fragment thereof(AB) that specifically binds to EGFR, wherein the AB is or is derivedfrom cetuximab; (ii) a masking moiety (MM) that inhibits the binding ofthe AB of the activatable antibody in an uncleaved state to EGFR; and(c) a cleavable moiety (CM) coupled to the AB, wherein the CM is apolypeptide that functions as a substrate for a protease.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to EGFR.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is no more than the equilibrium dissociationconstant of the AB to EGFR.

In some embodiments, the MM does not interfere or compete with the AB ofthe activatable antibody in a cleaved state for binding to EGFR.

In some embodiments, the protease is co-localized with EGFR in a tissue,and the protease cleaves the CM in the activatable antibody when theactivatable antibody is exposed to the protease. In some embodiments,the protease is not active or is significantly less active in tissuesthat do not significantly express EGFR. In some embodiments, theprotease is not active or is significantly less active in healthy, e.g.,non-diseased tissues.

In some embodiments, the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody includes a linking peptidebetween the MM and the CM.

In some embodiments, the activatable antibody includes a linking peptidebetween the CM and the AB.

In some embodiments, the activatable antibody includes a first linkingpeptide (LP1) and a second linking peptide (LP2), and wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides neednot be identical to each other.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to EGFR.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length, for example, no more than 40 amino acids long.

In some embodiments, the MM polypeptide sequence is different from thatof EGFR, and the MM polypeptide sequence is no more than 50% identicalto any natural binding partner of the AB.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 20-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 100-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 200-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind EGFR such that the dissociation constant (K_(d)) of the ABwhen coupled to the MM towards EGFR is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM towards EGFR. In someembodiments, the coupling of the MM reduces the ability of the AB tobind EGFR such that the K_(d) of the AB when coupled to the MM towardsEGFR is at least 100 times greater than the K_(d) of the AB when notcoupled to the MM towards EGFR. In some embodiments, the coupling of theMM reduces the ability of the AB to bind EGFR such that the K_(d) of theAB when coupled to the MM towards EGFR is at least 1000 times greaterthan the K_(d) of the AB when not coupled to the MM towards EGFR. Insome embodiments, the coupling of the MM reduces the ability of the ABto bind EGFR such that the K_(d) of the AB when coupled to the MMtowards EGFR is at least 10,000 times greater than the K_(d) of the ABwhen not coupled to the MM towards EGFR.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, in the presence of EGFR, the MM reduces the abilityof the AB to bind EGFR by at least 90% when the CM is uncleaved, ascompared to when the CM is cleaved when assayed in vitro using a targetdisplacement assay such as, for example, the assay described in PCTPublication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds EGFR. In someembodiments, the antibody or immunologically active fragment thereofthat binds EGFR is a monoclonal antibody, domain antibody, single chain,Fab fragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody. In someembodiments, such an antibody or immunologically active fragment thereofthat binds EGFR is a mouse, chimeric, humanized or fully humanmonoclonal antibody.

In some embodiments, the activatable antibody that in an activated statebinds Epidermal Growth Factor Receptor (EGFR) includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to EGFR,wherein the AB includes a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10,and a light chain amino acid that includes SEQ ID NO: 68; a maskingmoiety (MM) that inhibits the binding of the AB of the activatableantibody in an uncleaved state to EGFR, wherein the masking moietyincludes the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14); and acleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptidethat functions as a substrate for a protease, and wherein the CMincludes the amino acid sequence LSGRSDNH (SEQ ID NO: 13), wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM. In some embodiments, the AB of the activatable antibodyincludes a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid sequence including SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid sequence including SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acid sequenceincluding SEQ ID NO: 28. In some embodiments, the activatable antibodyincludes a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO: 10, and a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence including SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acid sequenceincluding SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO: 10, and a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence including SEQ ID NO: 4.

In some embodiments, the activatable antibody that in an activated statebinds Epidermal Growth Factor Receptor (EGFR) includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to EGFR,wherein the AB includes a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10,and a light chain amino acid that includes SEQ ID NO: 68; a maskingmoiety (MM) that inhibits the binding of the AB of the activatableantibody in an uncleaved state to EGFR, wherein the masking moietyincludes the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14); and acleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptidethat functions as a substrate for a protease, and wherein the CMincludes the amino acid sequence LSGRSDNH (SEQ ID NO: 13), wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM. In some embodiments, the AB of the activatable antibodyincludes a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid sequence including SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody is encoded by a nucleic acid sequenceencoding a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and anucleic acid encoding a light chain amino acid sequence including SEQ IDNO: 68. In some embodiments, the AB of the activatable antibody isencoded by a nucleic acid sequence encoding a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceincluding SEQ ID NO: 68.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10,and a nucleic acid encoding a light chain amino acid sequence includingSEQ ID NO: 28. In some embodiments, the activatable antibody is encodedby a nucleic acid sequence encoding a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceincluding SEQ ID NO: 28.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10,and a nucleic acid encoding a light chain amino acid sequence includingSEQ ID NO: 4. In some embodiments, the activatable antibody is encodedby a nucleic acid sequence encoding a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceincluding SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid including a heavy chain nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9,and a light chain nucleic acid sequence including SEQ ID NO: 67. In someembodiments, the AB of the activatable antibody is encoded by a nucleicacid sequence including a heavy chain nucleic acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acid sequence encodinga light chain nucleic acid that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence includingSEQ ID NO: 67.

In some embodiments, the activatable antibody is encoded by a nucleicacid including a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and alight chain nucleic acid sequence including SEQ ID NO: 27. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence including a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acid sequence encodinga light chain nucleic acid that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence includingSEQ ID NO: 27.

In some embodiments, the activatable antibody is encoded by a nucleicacid including a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and alight chain nucleic acid sequence including SEQ ID NO: 3. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence including a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acid sequence encodinga light chain nucleic acid that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence includingSEQ ID NO: 3.

In some embodiments, the AB of the activatable antibody includes a heavychain amino acid sequence selected from the group consisting of SEQ IDNO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acidsequence including SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody includes a heavy chain amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid sequence s including SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 26,SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acid sequenceincluding SEQ ID NO: 28. In some embodiments, the activatable antibodyincludes a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence s including SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 26,SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acid sequenceincluding SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence s including SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence encoding a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30, andSEQ ID NO: 34, and a nucleic acid sequence encoding a light chain aminoacid sequence including SEQ ID NO: 68. In some embodiments, the AB ofthe activatable antibody is encoded by a nucleic acid sequence thatincludes a nucleic acid sequence encoding a includes a heavy chain aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an amino acid selected from the group consistingof SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and a nucleic acidsequence encoding a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence including SEQ ID NO: 68.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34,and a nucleic acid sequence encoding a light chain amino acid sequenceincluding SEQ ID NO: 28. In some embodiments, the activatable antibodyis encoded by a nucleic acid sequence that includes a nucleic acidsequence encoding a includes a heavy chain amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toan amino acid including SEQ ID NO: 68, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceincluding SEQ ID NO: 28.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34,and a nucleic acid sequence encoding a light chain amino acid sequenceincluding SEQ ID NO: 4. In some embodiments, the activatable antibody isencoded by a nucleic acid sequence that includes a nucleic acid sequenceencoding a includes a heavy chain amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an aminoacid including SEQ ID NO: 68, and a nucleic acid sequence encoding alight chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence including SEQID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid including a heavy chain nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33,and a light chain nucleic acid sequence including SEQ ID NO: 67. In someembodiments, the AB of the activatable antibody is encoded by a nucleicacid sequence including a heavy chain nucleic acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceincluding SEQ ID NO: 67.

In some embodiments, the activatable antibody is encoded by a nucleicacid including a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, anda light chain nucleic acid sequence including SEQ ID NO: 27. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence including a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceincluding SEQ ID NO: 27.

In some embodiments, the activatable antibody is encoded by a nucleicacid including a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, anda light chain nucleic acid sequence including SEQ ID NO: 3. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence including a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceincluding SEQ ID NO: 3.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 26, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 26, and a light chain including an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 26, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 26, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 26, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 26, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 2, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 2, and a light chain including an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 2, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 2, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 2, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 2, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 6, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 6, and a light chain including an amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical tothe amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 6, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 6, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 6, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 6, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 30, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 30, and a light chain including an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 30, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 30, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 30, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 30, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 10, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 10, and a light chain including an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 10, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 10, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 10, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 10, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody includes a heavychain including the amino acid sequence of SEQ ID NO: 34, and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 34, and a light chain including an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 34, and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 34, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 28.

In some embodiments, the activatable antibody includes a heavy chainincluding the amino acid sequence of SEQ ID NO: 34, and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 34, and a lightchain including an amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequenceof SEQ ID NO: 4.

In some embodiments, the CM is a substrate for an enzyme selected fromthe group consisting of uPA, legumain and MT-SP1. In some embodiments,the enzyme comprises uPA. In some embodiments, the enzyme compriseslegumain. In some embodiments, the enzyme comprises MT-SP1.

In some embodiments, the CM includes the amino acid sequence LSGRSDNH(SEQ ID NO: 13).

In some embodiments, the MM does not include more than 25% amino acidsequence identity to EGFR. In some embodiments, the MM does not includemore than 10% amino acid sequence identity to EGFR.

In some embodiments, the MM includes the amino acid sequenceCISPRGCPDGPYVMY (SEQ ID NO: 14).

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 15) and (GGGS)_(n) (SEQ ID NO: 16), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19),GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22).

In some embodiments, LP1 includes the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 23).

In some embodiments, LP2 includes the amino acid sequence GSSGT (SEQ IDNO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the anti-EGFR activatable antibody is exposed toand cleaved by uPA or MT-SP1 such that, in the activated or cleavedstate, the activated antibody includes the light chain amino acidsequence of SEQ ID NO: 69. In some embodiments, the anti-EGFRactivatable antibody is exposed to and cleaved by legumain such that, inthe activated or cleaved state, the activated antibody includes thelight chain amino acid sequence of SEQ ID NO: 70.

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is an agent selected from the group listed inTable 1. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof.

In some embodiments, the agent is conjugated to the AB via a linker. Insome embodiments, the linker is a cleavable linker. In some embodiments,the linker is selected from the group consisting of the linkers shown inTables 2 and 3.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent. In some embodiments, the detectable moiety is a conjugatabledetection reagent. In some embodiments, the detectable moiety is, forexample, a fluorescein derivative such as fluorescein isothiocyanate(FITC).

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer includes atleast the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, an activatable antibody includes a spacer of sequenceQGQSGQ (SEQ ID NO: 38) joined directly to a MM sequence CISPRGCPDGPYVMY(SEQ ID NO: 14) in the structural arrangement from N-terminus toC-terminus of spacer-MM-CM-AB. In some embodiments, an activatableantibody includes a spacer joined directly to a MM sequence and includesthe amino acid sequence QGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 59) in thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.

In some embodiments, the serum half-life of the activatable antibody isat least 5 days when administered to an organism. In some embodiments,the serum half-life of the activatable antibody is at least 4 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 2 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least24 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 20 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 18 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least14 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 12 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 8 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 4 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 hours when administered to anorganism.

In some embodiments, the activatable anti-EGFR antibody is monospecific.In some embodiments, the activatable anti-EGFR antibody ismultispecific, e.g., by way of non-limiting example, bispecific ortrifunctional. In some embodiments, the activatable anti-EGFR antibodyis formulated as part of a pro-Bispecific T Cell Engager (BITE)molecule. In some embodiments, the activatable anti-EGFR antibody isformulated as part of a pro-Chimeric Antigen Receptor (CAR) modified Tcell or other engineered receptor.

In some embodiments, the activatable antibody that in an activated statebinds Epidermal Growth Factor Receptor (EGFR) includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to EGFR,wherein the AB includes a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34,and a light chain amino acid that includes SEQ ID NO: 68; a maskingmoiety (MM) that inhibits the binding of the AB of the activatableantibody in an uncleaved state to EGFR, wherein the masking moietyincludes the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14); and acleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptidethat functions as a substrate for a protease, and wherein the CMincludes the amino acid sequence LSGRSDNH (SEQ ID NO: 13), wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM. In some embodiments, the activatable antibody includes thelight chain amino acid sequence of SEQ ID NO: 68 and a spacer amino acidsequence. In some embodiments, the activatable antibody includes thelight chain amino acid of SEQ ID NO: 68 and a spacer amino acid sequencethat includes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody that in an activated statebinds Epidermal Growth Factor Receptor (EGFR) includes an antibody or anantigen binding fragment thereof (AB) that specifically binds to EGFR,wherein the AB includes a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10,and a light chain amino acid that includes SEQ ID NO: 68; a maskingmoiety (MM) that inhibits the binding of the AB of the activatableantibody in an uncleaved state to EGFR, wherein the masking moietyincludes the amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14); and acleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptidethat functions as a substrate for a protease, and wherein the CMincludes the amino acid sequence LSGRSDNH (SEQ ID NO: 13), wherein theactivatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM. In some embodiments, the activatable antibody includes thelight chain amino acid sequence of SEQ ID NO: 68 and a spacer amino acidsequence. In some embodiments, the activatable antibody includes thelight chain amino acid of SEQ ID NO: 68 and a spacer amino acid sequencethat includes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the antigen binding fragment thereof is selectedfrom the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,and a scAb.

In some embodiments, the AB of the activatable antibody includes acombination of heavy and light chains selected from the group consistingof: a heavy chain including amino acid sequence of SEQ ID NO: 2 and alight chain including the amino acid sequence of SEQ ID NO: 68; a heavychain including amino acid sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence of SEQ ID NO: 68; a heavy chainincluding amino acid sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence of SEQ ID NO: 68; a heavy chainincluding amino acid sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence of SEQ ID NO: 68; a heavy chainincluding amino acid sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence of SEQ ID NO: 68; and a heavy chainincluding amino acid sequence of SEQ ID NO: 34 and a light chainincluding the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain includingamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the sequence of SEQ ID NO: 2 and a lightchain including the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ IDNO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain includingamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the sequence of SEQ ID NO: 26 and a lightchain including the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ IDNO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain includingamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the sequence of SEQ ID NO: 6 and a lightchain including the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ IDNO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain includingamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the sequence of SEQ ID NO: 30 and a lightchain including the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ IDNO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the AB of the activatable antibody includes a heavy chain includingamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the sequence of SEQ ID NO: 10 and a lightchain including the amino acid sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ IDNO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB of the activatable antibody includes and aheavy chain including amino acid sequence of SEQ ID NO: 34 and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody includes a heavy chainincluding amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 34 anda light chain including the amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequenceof SEQ ID NO: 68.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 34 and a light chainincluding the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 34 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:4.

In some embodiments, the activatable antibody includes a heavy chainincluding amino acid sequence of SEQ ID NO: 34 and a light chainincluding the amino acid sequence of SEQ ID NO: 28. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the sequence of SEQ ID NO: 34 and a light chainincluding the amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:28.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to EGFR.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to EGFR. In some embodiments, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to EGFR.

In some embodiments, the MM is a polypeptide of no more than 40 aminoacids in length. In some embodiments, the MM polypeptide sequence isdifferent from that of EGFR, and the MM polypeptide sequence is no morethan 50% identical to any natural binding partner of the AB. In someembodiments, the MM does not include more than 25% amino acid sequenceidentity to EGFR. In some embodiments, the MM does not include more than10% amino acid sequence identity to EGFR.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength. In some embodiments, the CM is a substrate for an enzymeselected from the group consisting of uPA, legumain and MT-SP1. In someembodiments, the enzyme comprises uPA. In some embodiments, the enzymecomprises legumain. In some embodiments, the enzyme comprises MT-SP1. Insome embodiments, the protease is co-localized with EGFR in a tissue,and wherein the protease cleaves the CM in the activatable antibody whenthe activatable antibody is exposed to the protease.

In some embodiments, the activatable antibody includes a linking peptidebetween the MM and the CM. In some embodiments, the activatable antibodyincludes a linking peptide between the CM and the AB. In someembodiments, the activatable antibody includes a first linking peptide(LP1) and a second linking peptide (LP2), and the activatable antibodyhas the structural arrangement from N-terminus to C-terminus as followsin the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In someembodiments, the two linking peptides need not be identical to eachother.

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 15) and (GGGS)_(n) (SEQ ID NO: 16), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19),GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22).In some embodiments, LP1 includes the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 23). In some embodiments, LP2 includes the amino acidsequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the anti-EGFR activatable antibody is exposed toand cleaved by uPA or MT-SP1 such that, in the activated or cleavedstate, the activated antibody includes the light chain amino acidsequence of SEQ ID NO: 69. In some embodiments, the anti-EGFRactivatable antibody is exposed to and cleaved by legumain such that, inthe activated or cleaved state, the activated antibody includes thelight chain amino acid sequence of SEQ ID NO: 70.

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is an agent selected from the group listed inTable 1. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof.

In some embodiments, the agent is conjugated to the AB via a linker. Insome embodiments, the linker is a cleavable linker. In some embodiments,the linker is selected from the group consisting of the linkers shown inTables 2 and 3.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent. In some embodiments, the detectable moiety is a conjugatabledetection reagent. In some embodiments, the detectable moiety is, forexample, a fluorescein derivative such as fluorescein isothiocyanate(FITC).

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer includes atleast the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, an activatable antibody includes a spacer of sequenceQGQSGQ (SEQ ID NO: 38) joined directly to a MM sequence CISPRGCPDGPYVMY(SEQ ID NO: 14) in the structural arrangement from N-terminus toC-terminus of spacer-MM-CM-AB. In some embodiments, an activatableantibody includes a spacer joined directly to a MM sequence and includesthe amino acid sequence QGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 59) in thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.

In some embodiments, the serum half-life of the activatable antibody isat least 5 days when administered to an organism. In some embodiments,the serum half-life of the activatable antibody is at least 4 days whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 days when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 2 days when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least24 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 20 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 18 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least14 hours when administered to an organism. In some embodiments, theserum half-life of the activatable antibody is at least 12 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 10 hours when administered to anorganism. In some embodiments, the serum half-life of the activatableantibody is at least 8 hours when administered to an organism. In someembodiments, the serum half-life of the activatable antibody is at least6 hours when administered to an organism. In some embodiments, the serumhalf-life of the activatable antibody is at least 4 hours whenadministered to an organism. In some embodiments, the serum half-life ofthe activatable antibody is at least 3 hours when administered to anorganism.

In some embodiments, the activatable anti-EGFR antibody is monospecific.In some embodiments, the activatable anti-EGFR antibody ismultispecific, e.g., by way of non-limiting example, bispecific ortrifunctional. In some embodiments, the activatable anti-EGFR antibodyis formulated as part of a pro-Bispecific T Cell Engager (BITE)molecule. In some embodiments, the activatable anti-EGFR antibody isformulated as part of a pro-Chimeric Antigen Receptor (CAR) modified Tcell or other engineered receptor.

The invention also provides conjugated activatable antibodies that bindEpidermal Growth Factor Receptor (EGFR) in an activated state andinclude an antibody or an antigen binding fragment thereof (AB) thatspecifically binds to EGFR; a masking moiety (MM) that inhibits thebinding of the AB of the activatable antibody in an uncleaved state toEGFR; a cleavable moiety (CM) coupled to the AB, wherein the CM is apolypeptide that functions as a substrate for a protease, and acytotoxic agent.

In some embodiments, the AB is or is derived from cetuximab.

In some embodiments of the conjugated activatable antibodies, theantigen binding fragment thereof is selected from the group consistingof a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, a dAb, a singledomain heavy chain antibody, and a single domain light chain antibody.

In some embodiments, the AB of the conjugated activatable antibodyincludes a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and alight chain amino acid that includes SEQ ID NO: 68. In some embodiments,the AB of the conjugated activatable antibody includes a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6 and SEQ ID NO: 10, and a light chain amino acid thatincludes SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 2, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 26, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 6, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 30, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 10, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the AB of the conjugatedactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 34, and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the activatable antibodyincludes the light chain amino acid sequence of SEQ ID NO: 68 and aspacer amino acid sequence. In some embodiments, the activatableantibody includes the light chain amino acid of SEQ ID NO: 68 and aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesthe light chain amino acid sequence of SEQ ID NO: 68 joined directly toa spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38).

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 2 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 2 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain including amino acid sequence of SEQ ID NO: 2 anda light chain including the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 26 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 26 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain including amino acid sequence of SEQ ID NO: 26and a light chain including the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 6 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 6 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain including amino acid sequence of SEQ ID NO: 6 anda light chain including the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 30 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 30 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain including amino acid sequence of SEQ ID NO: 30and a light chain including the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the AB of the activatable antibody includes a heavychain including amino acid sequence of SEQ ID NO: 10 and a light chainincluding the amino acid sequence of SEQ ID NO: 68. In some embodiments,the activatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 10 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the activatable antibodyincludes a heavy chain including amino acid sequence of SEQ ID NO: 10and a light chain including the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the AB of the activatable antibody includes and aheavy chain including amino acid sequence of SEQ ID NO: 34 and a lightchain including the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes a heavy chain includingamino acid sequence of SEQ ID NO: 34 and a light chain including theamino acid sequence of SEQ ID NO: 4. In some embodiments, theactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 34 and a light chain including the amino acidsequence of SEQ ID NO: 28.

In some embodiments of the conjugated activatable antibody, the AB hasan equilibrium dissociation constant of about 100 nM or less for bindingto EGFR.

In some embodiments of the conjugated activatable antibody, the MM hasan equilibrium dissociation constant for binding to the AB which isgreater than the equilibrium dissociation constant of the AB to EGFR. Insome embodiments of the conjugated activatable antibody, the MM does notinterfere or compete with the AB of the activatable antibody in acleaved state for binding to EGFR. In some embodiments of the conjugatedactivatable antibody, the MM is a polypeptide of no more than 40 aminoacids in length. In some embodiments of the conjugated activatableantibody, the MM polypeptide sequence is different from that of EGFR,and the MM polypeptide sequence is no more than 50% identical to anynatural binding partner of the AB. In some embodiments of the conjugatedactivatable antibody, the MM does not include more than 25% amino acidsequence identity to EGFR. In some embodiments of the conjugatedactivatable antibody, the MM does not include more than 10% amino acidsequence identity to EGFR. In some embodiments of the conjugatedactivatable antibody, the MM includes the amino acid sequenceCISPRGCPDGPYVMY (SEQ ID NO: 14).

In some embodiments of the conjugated activatable antibody, the CM is apolypeptide of up to 15 amino acids in length. In some embodiments ofthe conjugated activatable antibody, the CM is a substrate for an enzymeselected from the group consisting of uPA, legumain and MT-SP1. In someembodiments, the enzyme comprises uPA. In some embodiments, the enzymecomprises legumain. In some embodiments, the enzyme comprises MT-SP1. Insome embodiments of the conjugated activatable antibody, the CM includesthe amino acid sequence LSGRSDNH (SEQ ID NO: 13).

In some embodiments, the conjugated activatable antibody includes alinking peptide between the MM and the CM. In some embodiments, theconjugated activatable antibody includes a linking peptide between theCM and the AB. In some embodiments, the conjugated activatable antibodyincludes a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the conjugated activatable antibody has thestructural arrangement from N-terminus to C-terminus as follows in theuncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In someembodiments, the two linking peptides need not be identical to eachother.

In some embodiments of the conjugated activatable antibody, at least oneof LP1 or LP2 includes an amino acid sequence selected from the groupconsisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 15) and(GGGS)_(n) (SEQ ID NO: 16), where n is an integer of at least one. Insome embodiments of the conjugated activatable antibody, at least one ofLP1 or LP2 includes an amino acid sequence selected from the groupconsisting of GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ IDNO: 19), GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), and GSSSG (SEQ IDNO: 22). In some embodiments of the conjugated activatable antibody, LP1includes the amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23). In someembodiments of the conjugated activatable antibody, LP2 includes theamino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the anti-EGFR activatable antibody is exposed toand cleaved by uPA or MT-SP1 such that, in the activated or cleavedstate, the activated antibody includes the light chain amino acidsequence of SEQ ID NO: 69. In some embodiments, the anti-EGFRactivatable antibody is exposed to and cleaved by legumain such that, inthe activated or cleaved state, the activated antibody includes thelight chain amino acid sequence of SEQ ID NO: 70.

In some embodiments, the cytotoxic agent included in the conjugatedactivatable antibody is an agent that is conjugated to the AB. In someembodiments of the conjugated activatable antibody, the agent is atherapeutic agent. In some embodiments, the agent is an antineoplasticagent. In some embodiments of the conjugated activatable antibody, theagent is a toxin or fragment thereof. In some embodiments of theconjugated activatable antibody, the agent is an agent selected from thegroup listed in Table 1. In some embodiments of the conjugatedactivatable antibody, the agent is a dolastatin. In some embodiments,the agent is an auristatin or derivative thereof. In some embodiments,the agent is auristatin E or a derivative thereof. In some embodimentsof the conjugated activatable antibody, the agent is monomethylauristatin E (MMAE). In some embodiments, the agent is a maytansinoid ormaytansinoid derivative. In some embodiments, the agent is DM1 or DM4.In some embodiments, the agent is a duocarmycin or derivative thereof.In some embodiments, the agent is a calicheamicin or derivative thereof.

In some embodiments of the conjugated activatable antibody, the agent isconjugated to the AB via a linker. In some embodiments of the conjugatedactivatable antibody, the linker is a cleavable linker. In someembodiments of the conjugated activatable antibody, the linker isselected from the group consisting of the linkers shown in Tables 2 and3.

In some embodiments, the conjugated activatable antibody also includes adetectable moiety. In some embodiments of the conjugated activatableantibody, the detectable moiety is a diagnostic agent. In someembodiments, the detectable moiety is a conjugatable detection reagent.In some embodiments, the detectable moiety is, for example, afluorescein derivative such as fluorescein isothiocyanate (FITC).

In some embodiments, the conjugated activatable antibody also includes asignal peptide. In some embodiments, the signal peptide is conjugated tothe conjugated activatable antibody via a spacer. In some embodiments,the spacer is conjugated to the conjugated activatable antibody in theabsence of a signal peptide. In some embodiments, the spacer is joineddirectly to the MM of the conjugated activatable antibody. In someembodiments, the spacer includes at least the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, an activatable antibody includes aspacer of sequence QGQSGQ (SEQ ID NO: 38) joined directly to a MMsequence CISPRGCPDGPYVMY (SEQ ID NO: 14) in the structural arrangementfrom N-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments,an activatable antibody includes a spacer joined directly to a MMsequence and includes the amino acid sequence QGQSGQCISPRGCPDGPYVMY (SEQID NO: 59) in the structural arrangement from N-terminus to C-terminusof spacer-MM-CM-AB.

In some embodiments, the serum half-life of the conjugated activatableantibody is at least 5 days when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 4 days when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 3 days when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 2 days when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 24 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 20 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 18 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 16 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 14 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 12 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 10 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 8 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 6 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 4 hours when administered to an organism. In someembodiments, the serum half-life of the conjugated activatable antibodyis at least 3 hours when administered to an organism.

In some embodiments, the conjugated activatable anti-EGFR antibody ismonospecific. In some embodiments, the conjugated activatable anti-EGFRantibody is multispecific, e.g., by way of non-limiting example,bispecific or trifunctional. In some embodiments, the conjugatedactivatable anti-EGFR antibody is formulated as part of a pro-BispecificT Cell Engager (BITE) molecule. In some embodiments, the conjugatedactivatable anti-EGFR antibody is formulated as part of a pro-ChimericAntigen Receptor (CAR) modified T cell or other engineered receptor.

The invention also provides an isolated nucleic acid molecule encodingan activatable anti-EGFR antibody described herein, as well as vectorsthat include these isolated nucleic acid sequences. The inventionprovides methods of producing an activatable antibody by culturing acell under conditions that lead to expression of the activatableantibody, wherein the cell includes such a vector.

In some embodiments, the nucleic acid encodes a MM that has anequilibrium dissociation constant for binding to the AB which is greaterthan the equilibrium dissociation constant of the AB to EGFR. In someembodiments, the nucleic acid encodes a MM that has an equilibriumdissociation constant for binding to the AB which is no more than theequilibrium dissociation constant of the AB to EGFR. In someembodiments, the nucleic acid encodes a MM that does not interfere orcompete with the AB of the activatable antibody in a cleaved state forbinding to EGFR.

In some embodiments, the nucleic acid encodes a CM that is cleaved by aprotease that is co-localized with EGFR in a tissue, and the proteasecleaves the CM in the activatable antibody when the activatable antibodyis exposed to the protease. In some embodiments, the protease is notactive or is significantly less active in tissues that do notsignificantly express EGFR. In some embodiments, the protease is notactive or is significantly less active in healthy, e.g., non-diseasedtissues.

In some embodiments, the nucleic acid encodes an activatable antibodythat has the structural arrangement from N-terminus to C-terminus asfollows in the uncleaved state: MM-CM-AB or AB-CM-MM.

In some embodiments, the nucleic acid encodes an activatable antibodythat includes a linking peptide between the MM and the CM.

In some embodiments, the nucleic acid encodes an activatable antibodythat includes a linking peptide between the CM and the AB.

In some embodiments, the nucleic acid encodes an activatable antibodythat includes a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the activatable antibody has the structuralarrangement from N-terminus to C-terminus as follows in the uncleavedstate: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, thetwo linking peptides need not be identical to each other.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 2 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 2 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 2 and a light chain including the amino acidsequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 26 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 26 and a light chain including the aminoacid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acidthat encodes an activatable antibody includes a heavy chain includingamino acid sequence of SEQ ID NO: 26 and a light chain including theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 6 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 6 and a light chain including the amino acidsequence of SEQ ID NO: 4. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 6 and a light chain including the amino acidsequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 30 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 30 and a light chain including the aminoacid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acidthat encodes an activatable antibody includes a heavy chain includingamino acid sequence of SEQ ID NO: 30 and a light chain including theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 10 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 10 and a light chain including the aminoacid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acidthat encodes an activatable antibody includes a heavy chain includingamino acid sequence of SEQ ID NO: 10 and a light chain including theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes the AB of anactivatable antibody includes a heavy chain including amino acidsequence of SEQ ID NO: 34 and a light chain including the amino acidsequence of SEQ ID NO: 68. In some embodiments, the nucleic acid thatencodes an activatable antibody includes a heavy chain including aminoacid sequence of SEQ ID NO: 34 and a light chain including the aminoacid sequence of SEQ ID NO: 4. In some embodiments, the nucleic acidthat encodes an activatable antibody includes a heavy chain includingamino acid sequence of SEQ ID NO: 34 and a light chain including theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 1 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 1 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 1and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 25 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 25 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 25and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 5 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 5 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 5and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 29 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 29 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 29and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 9 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 9 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 9and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain including nucleic acid sequence of SEQ ID NO: 33 and a lightchain including the nucleic acid sequence of SEQ ID NO: 67. In someembodiments, the nucleic acid includes a heavy chain including nucleicacid sequence of SEQ ID NO: 33 and a light chain including the nucleicacid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acidincludes a heavy chain including nucleic acid sequence of SEQ ID NO: 33and a light chain including the nucleic acid sequence of SEQ ID NO: 27.

In some embodiments, the nucleic acid encodes an AB that has anequilibrium dissociation constant of about 100 nM or less for binding toEGFR.

In some embodiments, the nucleic acid encodes a MM that is a polypeptideof about 2 to 40 amino acids in length, for example, no more than 40amino acids long.

In some embodiments, the nucleic acid encodes a MM polypeptide sequencethat is different from that of EGFR, and the MM polypeptide sequence isno more than 50% identical to any natural binding partner of the AB.

In some embodiments, the nucleic acid encodes a CM that is positioned inthe activatable antibody such that in the uncleaved state, binding ofthe activatable antibody to EGFR is reduced to occur with an equilibriumdissociation constant that is at least 20-fold greater than theequilibrium dissociation constant of an unmodified AB binding to EGFR,and whereas the AB of the activatable antibody in the cleaved statebinds EGFR.

In some embodiments, the nucleic acid encodes a CM that is positioned inthe activatable antibody such that in the uncleaved state, binding ofthe activatable antibody to EGFR is reduced to occur with an equilibriumdissociation constant that is at least 100-fold greater than theequilibrium dissociation constant of an unmodified AB binding to EGFR,and whereas the AB of the activatable antibody in the cleaved statebinds EGFR.

In some embodiments, the nucleic acid encodes a CM that is positioned inthe activatable antibody such that in the uncleaved state, binding ofthe activatable antibody to EGFR is reduced to occur with an equilibriumdissociation constant that is at least 200-fold greater than theequilibrium dissociation constant of an unmodified AB binding to EGFR,and whereas the AB of the activatable antibody in the cleaved statebinds EGFR.

In some embodiments, the nucleic acid encodes a CM that is a polypeptideof up to 15 amino acids in length.

In some embodiments, nucleic acid encodes a MM, that in the presence ofEGFR, reduces the ability of the AB to bind EGFR by at least 90% whenthe CM is uncleaved, as compared to when the CM is cleaved when assayedin vitro using a target displacement assay such as, for example, theassay described in PCT Publication Nos. WO 2009/025846 and WO2010/081173.

In some embodiments, the nucleic acid encodes an activatable antibodythat includes an antibody or antigen-binding fragment thereof thatspecifically binds EGFR. In some embodiments, the nucleic acid encodesan antibody or immunologically active fragment thereof that binds EGFRand is a monoclonal antibody, domain antibody, single chain, Fabfragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody. In someembodiments, the nucleic acid encodes an antibody or immunologicallyactive fragment thereof that binds EGFR and is a mouse, chimeric,humanized or fully human monoclonal antibody.

In some embodiments, the nucleic acid encodes the AB of an activatableantibody that includes a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10,and a light chain amino acid sequence including SEQ ID NO: 68. In someembodiments, the nucleic acid encodes the AB of an activatable antibodythat includes a heavy chain amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO: 10, and a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence including SEQ ID NO: 68. In some embodiments, the activatableantibody includes the light chain amino acid sequence of SEQ ID NO: 68and a spacer amino acid sequence. In some embodiments, the activatableantibody includes the light chain amino acid of SEQ ID NO: 68 and aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid sequence ofSEQ ID NO: 68 joined directly to a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid of SEQ ID NO: 68 and a spaceramino acid sequence that includes the amino acid sequence QGQSGQ (SEQ IDNO: 38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid sequence of SEQID NO: 68 joined directly to a spacer amino acid sequence that includesthe amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the nucleic acid encodes an activatable antibodythat includes a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid sequence including SEQ ID NO: 28. In some embodiments,the nucleic acid encodes an activatable antibody that includes a heavychain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to an amino acid sequence including SEQ ID NO:28.

In some embodiments, the nucleic acid encodes an activatable antibodythat includes a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid sequence including SEQ ID NO: 4. In some embodiments,the nucleic acid encodes an activatable antibody that includes a heavychain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to an amino acid sequence including SEQ ID NO:4.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and a light chain nucleicacid sequence that includes SEQ ID NO: 67. In some embodiments, thenucleic acid sequence includes an AB that includes a heavy chain nucleicacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and a lightchain nucleic acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to a nucleic acid sequence including SEQ IDNO: 67.

In some embodiments, the nucleic acid includes a heavy chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 5, and SEQ ID NO: 9, and a light chain nucleic acid sequence thatincludes SEQ ID NO: 27. In some embodiments, the nucleic acid sequenceincludes a heavy chain nucleic acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, and SEQ ID NO: 9, and a light chain nucleic acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anucleic acid sequence including SEQ ID NO: 27.

In some embodiments, the nucleic acid includes a heavy chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 5, and SEQ ID NO: 9, and a light chain nucleic acid sequence thatincludes SEQ ID NO: 3. In some embodiments, the nucleic acid sequenceincludes a heavy chain nucleic acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an nucleic acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, and SEQ ID NO: 9, and a light chain nucleic acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anucleic acid sequence including SEQ ID NO: 3.

In some embodiments, the nucleic acid that encodes an activatableantibody includes an AB that includes a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30 andSEQ ID NO: 34, and a light chain amino acid sequence including SEQ IDNO: 68. In some embodiments, the activatable antibody includes an ABthat includes a heavy chain amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence s including SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the nucleic acid that encodes an activatableantibody includes a heavy chain amino acid sequence selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, anda light chain amino acid sequence including SEQ ID NO: 28. In someembodiments, the activatable antibody includes a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and alight chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence sincluding SEQ ID NO: 28.

In some embodiments, the nucleic acid that encodes an activatableantibody includes a heavy chain amino acid sequence selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, anda light chain amino acid sequence including SEQ ID NO: 4. In someembodiments, the activatable antibody includes a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and alight chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence sincluding SEQ ID NO: 4.

In some embodiments, the nucleic acid includes an AB that includes aheavy chain nucleic acid sequence selected from the group consisting ofSEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, and a light chainnucleic acid sequence that includes SEQ ID NO: 67. In some embodiments,the nucleic acid sequence includes an AB that includes a heavy chainnucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an nucleic acid sequence selected fromthe group consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33,and a light chain nucleic acid that is at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence includingSEQ ID NO: 67.

In some embodiments, the nucleic acid includes a heavy chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 25, SEQID NO: 29, and SEQ ID NO: 33, and a light chain nucleic acid sequencethat includes SEQ ID NO: 27. In some embodiments, the nucleic acidsequence includes a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a light chain nucleic acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to a nucleic acid sequence including SEQ ID NO: 27.

In some embodiments, the nucleic acid includes a heavy chain nucleicacid sequence selected from the group consisting of SEQ ID NO: 25, SEQID NO: 29, and SEQ ID NO: 33, and a light chain nucleic acid sequencethat includes SEQ ID NO: 4. In some embodiments, the nucleic acidsequence includes a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a light chain nucleic acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to a nucleic acid sequence including SEQ ID NO: 4.

In some embodiments, the nucleic acid encodes a CM that is a substratefor an enzyme selected from the group consisting of uPA, legumain andMT-SP1. In some embodiments, the enzyme comprises uPA. In someembodiments, the enzyme comprises legumain. In some embodiments, theenzyme comprises MT-SP1.

In some embodiments, the nucleic acid encodes a CM that includes theamino acid sequence LSGRSDNH (SEQ ID NO: 13).

In some embodiments, the nucleic acid encodes a MM that does not includemore than 25% amino acid sequence identity to EGFR. In some embodiments,the nucleic acid encodes a MM that does not include more than 10% aminoacid sequence identity to EGFR.

In some embodiments, the nucleic acid encodes a MM that includes theamino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 14).

In some embodiments, the nucleic acid encodes an LP1 and/or LP2, whereinto least one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 15)and (GGGS)_(n) (SEQ ID NO: 16), where n is an integer of at least one.

In some embodiments, the nucleic acid encodes an LP1 and/or LP2, whereinat least one of LP1 or LP2 includes an amino acid sequence selected fromthe group consisting of GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18),GSGSG (SEQ ID NO: 19), GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), andGSSSG (SEQ ID NO: 22).

In some embodiments, the nucleic acid encodes an LP1 that includes theamino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 23).

In some embodiments, the nucleic acid encodes an LP2 that includes theamino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the nucleic acid encodes an activatable antibodythat also includes a signal peptide. In some embodiments, the nucleicacid encodes a signal peptide that is conjugated to the activatableantibody via a spacer. In some embodiments, the nucleic acid encodes aspacer that is conjugated to the activatable antibody in the absence ofa signal peptide. In some embodiments, the nucleic acid encodes a spacerthat is joined directly to the MM of the activatable antibody. In someembodiments, the nucleic acid encodes a spacer that includes at leastthe amino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, thenucleic acid encodes an activatable antibody that includes a spacer ofsequence QGQSGQ (SEQ ID NO: 38) joined directly to a MM sequenceCISPRGCPDGPYVMY (SEQ ID NO: 14) in the structural arrangement fromN-terminus to C-terminus of spacer-MM-CM-AB. In some embodiments, thenucleic acid encodes an activatable antibody that includes a spacerjoined directly to a MM sequence and includes the amino acid sequenceQGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 59) in the structural arrangement fromN-terminus to C-terminus of spacer-MM-CM-AB.

The invention also provides a method of manufacturing activatableantibodies that in an activated state binds Epidermal Growth FactorReceptor (EGFR) by (a) culturing a cell comprising a nucleic acidconstruct that encodes the activatable antibody under conditions thatlead to expression of the activatable antibody, wherein the activatableantibody comprises a masking moiety (MM), a cleavable moiety (CM), andan antibody or an antigen binding fragment thereof (AB) thatspecifically binds EGFR, (i) wherein the CM is a polypeptide thatfunctions as a substrate for a protease; and (ii) wherein the CM ispositioned in the activatable antibody such that, in an uncleaved state,the MM interferes with specific binding of the AB to EGFR and, when theactivatable antibody is in a cleaved state, the MM does not interfere orcompete with specific binding of the AB to EGFR; and (b) recovering theactivatable antibody.

In some embodiments, the AB is or is derived from cetuximab,

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to EGFR.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is no more than the equilibrium dissociationconstant of the AB to EGFR.

In some embodiments, the MM does not interfere or compete with the AB ofthe activatable antibody in a cleaved state for binding to EGFR.

In some embodiments, the protease is co-localized with EGFR in a tissue,and the protease cleaves the CM in the activatable antibody when theactivatable antibody is exposed to the protease. In some embodiments,the protease is not active or is significantly less active in tissuesthat do not significantly express EGFR. In some embodiments, theprotease is not active or is significantly less active in healthy, e.g.,non-diseased tissues.

In some embodiments, the activatable antibody has the structuralarrangement from N-terminus to C-terminus as follows in the uncleavedstate: MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the MM and the CM.

In some embodiments, the activatable antibody comprises a linkingpeptide between the CM and the AB.

In some embodiments, the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2), and wherein theactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: MM-LP1-CM-LP2-AB orAB-LP2-CM-LP1-MM.

In some embodiments, the two linking peptides need not be identical toeach other.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to EGFR.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length, for example, no more than 40 amino acids long.

In some embodiments, the MM polypeptide sequence is different from thatof EGFR, and the MM polypeptide sequence is no more than 50% identicalto any natural binding partner of the AB.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 20-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 100-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodysuch that in the uncleaved state, binding of the activatable antibody toEGFR is reduced to occur with an equilibrium dissociation constant thatis at least 200-fold greater than the equilibrium dissociation constantof an unmodified AB binding to EGFR, and whereas the AB of theactivatable antibody in the cleaved state binds EGFR.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind EGFR such that the dissociation constant (K_(d)) of the ABwhen coupled to the MM towards EGFR is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM towards EGFR. In someembodiments, the coupling of the MM reduces the ability of the AB tobind EGFR such that the K_(d) of the AB when coupled to the MM towardsEGFR is at least 100 times greater than the K_(d) of the AB when notcoupled to the MM towards EGFR. In some embodiments, the coupling of theMM reduces the ability of the AB to bind EGFR such that the K_(d) of theAB when coupled to the MM towards EGFR is at least 1000 times greaterthan the K_(d) of the AB when not coupled to the MM towards EGFR. Insome embodiments, the coupling of the MM reduces the ability of the ABto bind EGFR such that the K_(d) of the AB when coupled to the MMtowards EGFR is at least 10,000 times greater than the K_(d) of the ABwhen not coupled to the MM towards EGFR.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, in the presence of EGFR, the MM reduces the abilityof the AB to bind EGFR by at least 90% when the CM is uncleaved, ascompared to when the CM is cleaved when assayed in vitro using a targetdisplacement assay such as, for example, the assay described in PCTPublication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the activatable antibody includes an antibody orantigen-binding fragment thereof that specifically binds EGFR. In someembodiments, the antibody or immunologically active fragment thereofthat binds EGFR is a monoclonal antibody, domain antibody, single chain,Fab fragment, a F(ab′)₂ fragment, a scFv, a scab, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody. In someembodiments, such an antibody or immunologically active fragment thereofthat binds EGFR is a mouse, chimeric, humanized or fully humanmonoclonal antibody.

In some embodiments, the AB of the activatable antibody comprises aheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain aminoacid sequence comprising SEQ ID NO: 68. In some embodiments, the AB ofthe activatable antibody comprises a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid sequence comprising SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the activatable antibody comprises a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acid sequencecomprising SEQ ID NO: 28. In some embodiments, the activatable antibodycomprises a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO: 10, and a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence comprising SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acid sequencecomprising SEQ ID NO: 4. In some embodiments, the activatable antibodycomprises a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, andSEQ ID NO: 10, and a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence comprising SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence that comprises a nucleic acid sequence encoding aheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acidencoding a light chain amino acid sequence comprising SEQ ID NO: 68. Insome embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence encoding a comprises a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequencecomprising SEQ ID NO: 68. In some embodiments, the activatable antibodyincludes the light chain amino acid sequence of SEQ ID NO: 68 and aspacer amino acid sequence. In some embodiments, the activatableantibody includes the light chain amino acid of SEQ ID NO: 68 and aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid sequence ofSEQ ID NO: 68 joined directly to a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid of SEQ ID NO: 68 and a spaceramino acid sequence that includes the amino acid sequence QGQSGQ (SEQ IDNO: 38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid sequence of SEQID NO: 68 joined directly to a spacer amino acid sequence that includesthe amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence that comprises a nucleic acid sequence encoding aheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acidencoding a light chain amino acid sequence comprising SEQ ID NO: 67. Insome embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence encoding a comprises a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequencecomprising SEQ ID NO: 67.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence that comprises a nucleic acid sequence encoding a heavychain amino acid sequence selected from the group consisting of SEQ IDNO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a nucleic acid encoding alight chain amino acid sequence comprising SEQ ID NO: 4. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence encoding a comprises a heavy chain amino acid sequence that isat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical toan amino acid selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 6, and SEQ ID NO: 10, and a nucleic acid sequence encoding a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to an amino acid sequence comprising SEQ IDNO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid comprising a heavy chain nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO:9, and a light chain nucleic acid sequence comprising SEQ ID NO: 67. Insome embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence comprising a heavy chain nucleic acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acidsequence that comprises a nucleic acid sequence encoding a light chainnucleic acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to a nucleic acid sequence selected comprising SEQID NO: 67.

In some embodiments, the activatable antibody is encoded by a nucleicacid comprising a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and alight chain nucleic acid sequence comprising SEQ ID NO: 27. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence comprising a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acid sequence thatcomprises a nucleic acid sequence encoding a light chain nucleic acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to a nucleic acid sequence selected comprising SEQ ID NO: 27.

In some embodiments, the activatable antibody is encoded by a nucleicacid comprising a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and alight chain nucleic acid sequence comprising SEQ ID NO: 4. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence comprising a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acid sequence thatcomprises a nucleic acid sequence encoding a light chain nucleic acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to a nucleic acid sequence selected comprising SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain amino acid sequence selected from the group consisting ofSEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain aminoacid sequence comprising SEQ ID NO: 68. In some embodiments, the AB ofthe activatable antibody comprises a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid sequence selected from the group consistingof SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and a light chainamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to an amino acid sequence comprising SEQ IDNO: 68. In some embodiments, the activatable antibody includes the lightchain amino acid sequence of SEQ ID NO: 68 and a spacer amino acidsequence. In some embodiments, the activatable antibody includes thelight chain amino acid of SEQ ID NO: 68 and a spacer amino acid sequencethat includes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid sequence of SEQ ID NO: 68 joineddirectly to a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid of SEQ ID NO: 68 and a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid sequence of SEQ ID NO: 68 joineddirectly to a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 26,SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acid sequencecomprising SEQ ID NO: 28. In some embodiments, the activatable antibodycomprises a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence comprising SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 26,SEQ ID NO: 30 and SEQ ID NO: 34, and a light chain amino acid sequencecomprising SEQ ID NO: 4. In some embodiments, the activatable antibodycomprises a heavy chain amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anamino acid sequence comprising SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid sequence encoding a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 30, andSEQ ID NO: 34, and a nucleic acid sequence encoding a light chain aminoacid sequence comprising SEQ ID NO: 68. In some embodiments, the AB ofthe activatable antibody is encoded by a nucleic acid encoding a heavychain amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and anucleic acid sequence encoding a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an aminoacid sequence comprising SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34,and a nucleic acid sequence encoding a light chain amino acid sequencecomprising SEQ ID NO: 28. In some embodiments, the activatable antibodyis encoded by a nucleic acid encoding a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid selected from the group consisting of SEQ IDNO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequencecomprising SEQ ID NO: 68.

In some embodiments, the activatable antibody is encoded by a nucleicacid sequence encoding a heavy chain amino acid sequence selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34,and a nucleic acid sequence encoding a light chain amino acid sequencecomprising SEQ ID NO: 4. In some embodiments, the activatable antibodyis encoded by a nucleic acid encoding a heavy chain amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid selected from the group consisting of SEQ IDNO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequencecomprising SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody is encoded by anucleic acid comprising a heavy chain nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ IDNO: 33, and a light chain nucleic acid sequence comprising SEQ ID NO:67. In some embodiments, the AB of the activatable antibody is encodedby a nucleic acid sequence comprising a heavy chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, and anucleic acid sequence encoding a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence comprising SEQ ID NO: 67.

In some embodiments, the activatable antibody is encoded by a nucleicacid comprising a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, anda light chain nucleic acid sequence comprising SEQ ID NO: 27. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence comprising a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an nucleic acid sequencecomprising SEQ ID NO: 27.

In some embodiments, the activatable antibody is encoded by a nucleicacid comprising a heavy chain nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, anda light chain nucleic acid sequence comprising SEQ ID NO: 3. In someembodiments, the activatable antibody is encoded by a nucleic acidsequence comprising a heavy chain nucleic acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to annucleic acid sequence selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 29, and SEQ ID NO: 33, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an nucleic acid sequencecomprising SEQ ID NO: 3.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 2, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 2, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:2, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 26, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 26, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 26, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:26, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 26, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 26, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 6, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 6, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 6, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 6, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 6, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 30, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 30, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 30, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:30, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 30, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 30, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 10, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 10, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 10, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:10, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 10, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 10, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the AB of the activatable antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 34, and alight chain comprising the amino acid sequence of SEQ ID NO: 68. In someembodiments, the AB of the activatable antibody comprises a heavy chaincomprising an amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence ofSEQ ID NO: 34, and a light chain comprising an amino acid sequence thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO: 68. In some embodiments, theactivatable antibody includes the light chain amino acid sequence of SEQID NO: 68 and a spacer amino acid sequence. In some embodiments, theactivatable antibody includes the light chain amino acid of SEQ ID NO:68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid of SEQ ID NO: 68and a spacer amino acid sequence that includes the amino acid sequenceQGQSGQ (SEQ ID NO: 38). In some embodiments, the activatable antibodyincludes a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to the light chain amino acidsequence of SEQ ID NO: 68 joined directly to a spacer amino acidsequence that includes the amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34, and a light chaincomprising the amino acid sequence of SEQ ID NO: 28. In someembodiments, the activatable antibody comprises a heavy chain comprisingan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:34, and a light chain comprising an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments, the activatable antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34, and a light chaincomprising the amino acid sequence of SEQ ID NO: 4. In some embodiments,the activatable antibody comprises a heavy chain comprising an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the amino acid sequence of SEQ ID NO: 34, and alight chain comprising an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 4.

In some embodiments, the CM is a substrate for an enzyme selected fromthe group consisting of uPA, legumain and MT-SP1. In some embodiments,the enzyme comprises uPA. In some embodiments, the enzyme compriseslegumain. In some embodiments, the enzyme comprises MT-SP1.

In some embodiments, the CM comprises the amino acid sequence LSGRSDNH(SEQ ID NO: 13).

In some embodiments, the MM does not comprise more than 25% amino acidsequence identity to EGFR.

In some embodiments, the MM does not comprise more than 10% amino acidsequence identity to EGFR.

In some embodiments, the MM comprises the amino acid sequenceCISPRGCPDGPYVMY (SEQ ID NO: 14).

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 15) and (GGGS)_(n) (SEQ ID NO: 16), where n isan integer of at least one.

In some embodiments, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 17),GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19), GSGGG (SEQ ID NO: 20),GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22).

In some embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 23).

In some embodiments, LP2 comprises the amino acid sequence GSSGT (SEQ IDNO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is conjugated to the AB via a linker. In someembodiments, the linker is a cleavable linker.

In some embodiments, the activatable antibody also includes a detectablemoiety. In some embodiments, the detectable moiety is a diagnosticagent. In some embodiments, the detectable moiety is a conjugatabledetection reagent. In some embodiments, the detectable moiety is, forexample, a fluorescein derivative such as fluorescein isothiocyanate(FITC).

In some embodiments, the activatable antibody also includes a signalpeptide. In some embodiments, the signal peptide is conjugated to theactivatable antibody via a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer includes atleast the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, an activatable antibody comprises a spacer of sequenceQGQSGQ (SEQ ID NO: 38) joined directly to a MM sequence CISPRGCPDGPYVMY(SEQ ID NO: 14) in the structural arrangement from N-terminus toC-terminus of spacer-MM-CM-AB. In some embodiments, an activatableantibody includes a spacer of sequence: joined directly to a MM sequenceand includes the amino acid sequence QGQSGQCISPRGCPDGPYVMY (SEQ ID NO:59) in the structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB.

The invention provides methods of treating, preventing and/or delayingthe onset or progression of, or alleviating a symptom of an indication,e.g., disease or disorder, associated with aberrant expression and/oractivity of EGFR, e.g., an EGFR-related disorder or EGFR-relateddisease, in a subject using a therapeutic molecule, e.g., activatableantibodies that bind EGFR and/or conjugated activatable antibodies thatbind EGFR, particularly activatable antibodies and/or conjugatedactivatable antibodies that bind and neutralize or otherwise inhibit atleast one biological activity of EGFR and/or EGFR-mediated signaling.

In some embodiments, the indication, e.g., disease or disorder,associated with aberrant expression and/or activity of EGFR is a cancer.In some embodiments, the cancer is a breast cancer, e.g., by way ofnon-limiting example, the breast cancer is a triple-negative breastcancer. In some embodiments, the cancer is a triple-negative breastcancer. In some embodiments, the cancer is colorectal cancer. In someembodiments, the cancer is gastric cancer. In some embodiments, thecancer is glioblastoma. In some embodiments, the cancer is a head andneck cancer, e.g., by way of non-limiting example, esophageal cancer. Insome embodiments, the cancer is an esophageal cancer. In someembodiments, the cancer is a lung cancer, e.g., by way of non-limitingexample, non-small cell lung cancer. In some embodiments, the cancer isa non-small cell lung cancer. In some embodiments, the cancer isovarian/endometrial cancer. In some embodiments, the cancer ispancreatic cancer. In some embodiments, the cancer is prostate cancer.In some embodiments, the cancer is a renal cancer. In some embodiments,the cancer is a sarcoma, e.g., by way of non-limiting example,osteosarcoma. In some embodiments, the cancer is an osteosarcoma. Insome embodiments, the cancer is a skin cancer, e.g., by way ofnon-limiting example, squamous cell cancer, basal cell carcinoma, and/ormelanoma. In some embodiments, the cancer is a squamous cell cancer. Insome embodiments, the cancer is a basal cell carcinoma. In someembodiments, the cancer is a melanoma.

In some embodiments, the indication, e.g., disease or disorder,associated with aberrant expression and/or activity of EGFR is aninflammatory disorder and/or an autoimmune disease. In some embodiments,the inflammatory and/or autoimmune disease is psoriasis.

The therapeutic molecule, e.g., activatable anti-EGFR antibody and/orconjugated anti-EGFR activatable antibody, can be administered at anystage of the disease. For example, a therapeutic molecule can beadministered to a patient suffering cancer of any stage, from early tometastatic. For example, therapeutic molecule can be administered to apatient suffering from an inflammatory disorder and/or autoimmunedisease of any stage, from early onset to an advanced stage. It is to beunderstood that the terms subject and patient are used interchangeablyherein.

The therapeutic molecules, e.g. activatable anti-EGFR antibodies and/orconjugated activatable anti-EGFR antibodies, are also useful in othertherapeutic indications and treatment regimens. For example, thetherapeutic molecules of the embodiments provided herein can be used ina treatment regimen that includes neoadjuvant therapy.

In some embodiments, the therapeutic molecules, e.g. activatableanti-EGFR antibodies and/or conjugated activatable anti-EGFR antibodies,are administered during and/or after treatment in combination with oneor more additional agents such as, by way of non-limiting example, achemotherapeutic agent. In some embodiments, the therapeutic moleculeand the additional agent(s) are administered simultaneously. Forexample, the therapeutic molecule and the additional agent(s) can beformulated in a single composition or administered as two or moreseparate compositions. In some embodiments, the therapeutic molecule andthe additional agent(s) are administered sequentially.

The invention also provides methods of preventing, delaying theprogression of, treating, alleviating a symptom of, or otherwiseameliorating an EGFR-related disorder, for example, cancer, in a subjectby administering a therapeutically effective amount of an activatableanti-EGFR antibody described herein to a subject in need thereof.

The invention also provides methods of inhibiting angiogenesis in asubject by administering a therapeutically effective amount of anactivatable anti-EGFR antibody described herein to a subject in needthereof.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human. In some embodiments, the subject is a non-humanmammal, such as a non-human primate, companion animal (e.g., cat, dog,horse), farm animal, work animal, or zoo animal.

The activatable anti-EGFR antibody and therapeutic formulations thereofare administered to a subject suffering from or susceptible to a diseaseor disorder associated with aberrant EGFR expression and/or activity. Asubject suffering from or susceptible to a disease or disorderassociated with aberrant EGFR expression and/or activity is identifiedusing any of a variety of methods known in the art. For example,subjects suffering from cancer or other neoplastic condition areidentified using any of a variety of clinical and/or laboratory testssuch as, physical examination and blood, urine and stool analysis toevaluate health status.

Administration of an activatable anti-EGFR antibody to a patientsuffering from a disease or disorder associated with aberrant EGFRexpression and/or activity is considered successful if any of a varietyof laboratory or clinical objectives is achieved. For example,administration of an activatable anti-EGFR antibody to a patientsuffering from a disease or disorder associated with aberrant EGFRexpression and/or activity is considered successful if one or more ofthe symptoms associated with the disease or disorder is alleviated,reduced, inhibited or does not progress to a further, i.e., worse,state. Administration of an activatable anti-EGFR antibody to a patientsuffering from a disease or disorder associated with aberrant EGFRexpression and/or activity is considered successful if the diabetesenters remission or does not progress to a further, i.e., worse, state.

In some embodiments, the activatable anti-EGFR antibody is administeredduring and/or after treatment in combination with one or more additionalagents such as, for example, a chemotherapeutic agent. In someembodiments, the activatable anti-EGFR antibody and the additionalagent(s) are administered simultaneously. For example, the activatableanti-EGFR antibody and the additional agent(s) can be formulated in asingle composition or administered as two or more separate compositions.In some embodiments, the activatable anti-EGFR antibody and theadditional agent(s) are administered sequentially.

The invention also provides methods of using activatable antibodies thatbind EGFR (i.e., activatable anti-EGFR antibodies, also referred toherein as anti-EGFR activatable antibodies) in a variety of diagnosticand/or prophylactic indications, as well as kits for use in thesemethods. For example, the invention provides methods of detectingpresence or absence of a cleaving agent and EGFR in a subject or asample by (i) contacting a subject or sample with an anti-EGFRactivatable antibody, and (ii) measuring a level of activated anti-EGFRactivatable antibody in the subject or sample, wherein a detectablelevel of activated anti-EGFR activatable antibody in the subject orsample indicates that the cleaving agent and EGFR are present in thesubject or sample and wherein no detectable level of activated anti-EGFRactivatable antibody in the subject or sample indicates that thecleaving agent, EGFR or both the cleaving agent and EGFR are absent inthe subject or sample. Such an anti-EGFR activatable antibody includes amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds EGFR, wherein the anti-EGFR activatable antibodyin an uncleaved (i.e., non-activated) state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (ii) wherein the MM is a peptide that inhibits binding of theAB to EGFR, and wherein the MM does not have an amino acid sequence of anaturally occurring binding partner of the AB; and (iii) wherein the MMof the activatable antibody in an uncleaved state interferes withspecific binding of the AB to EGFR, and wherein the MM of an activatableantibody in a cleaved (i.e., activated) state does not interfere orcompete with specific binding of the AB to EGFR.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a target of interest (EGFR)in a subject or a sample, where the kits include at least an anti-EGFRactivatable antibody and/or conjugated anti-EGFR activatable antibodydescribed herein for use in contacting a subject or sample and means fordetecting the level of activated anti-EGFR activatable antibody and/orconjugated anti-EGFR activatable antibody in the subject or sample,wherein a detectable level of activated anti-EGFR activatable antibodyin the subject or sample indicates that the cleaving agent and EGFR arepresent in the subject or sample and wherein no detectable level ofactivated anti-EGFR activatable antibody in the subject or sampleindicates that the cleaving agent, EGFR or both the cleaving agent andEGFR are absent in the subject or sample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an anti-EGFR activatable antibody in the presence of EGFR,and (ii) measuring a level of activated anti-EGFR activatable antibodyin the subject or sample, wherein a detectable level of activatedanti-EGFR activatable antibody in the subject or sample indicates thatthe cleaving agent is present in the subject or sample and wherein nodetectable level of activated anti-EGFR activatable antibody in thesubject or sample indicates that the cleaving agent is absent in thesubject or sample. Such an anti-EGFR activatable antibody includes amasking moiety (MM), a cleavable moiety (CM) that is cleaved by thecleaving agent, and an antigen binding domain or fragment thereof (AB)that specifically binds EGFR, wherein the anti-EGFR activatable antibodyin an uncleaved (i.e., non-activated) state comprises a structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM; (ii) wherein the MM is a peptide that inhibits binding of theAB to EGFR, and wherein the MM does not have an amino acid sequence of anaturally occurring binding partner of the AB; and (iii) wherein the MMof the activatable antibody in an uncleaved state interferes withspecific binding of the AB to EGFR, and wherein the MM of an activatableantibody in a cleaved (i.e., activated) state does not interfere orcompete with specific binding of the AB to EGFR.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent in a subject or a sample, wherethe kits include at least an anti-EGFR activatable antibody and/orconjugated anti-EGFR activatable antibody described herein for use incontacting a subject or sample and means for detecting the level ofactivated anti-EGFR activatable antibody and/or conjugated anti-EGFRactivatable antibody in the subject or sample, wherein a detectablelevel of activated anti-EGFR activatable antibody in the subject orsample indicates that the cleaving agent is present in the subject orsample and wherein no detectable level of activated anti-EGFRactivatable antibody in the subject or sample indicates that thecleaving agent is absent in the subject or sample.

The invention provides methods of detecting presence or absence of acleaving agent and EGFR in a subject or a sample by (i) contacting asubject or sample with an anti-EGFR activatable antibody, wherein theanti-EGFR activatable antibody includes a detectable label that ispositioned on a portion of the anti-EGFR activatable antibody that isreleased following cleavage of the CM and (ii) measuring a level ofactivated anti-EGFR activatable antibody in the subject or sample,wherein a detectable level of activated anti-EGFR activatable antibodyin the subject or sample indicates that the cleaving agent and EGFR arepresent in the subject or sample and wherein no detectable level ofactivated anti-EGFR activatable antibody in the subject or sampleindicates that the cleaving agent, EGFR or both the cleaving agent andEGFR are absent in the subject or sample. Such an anti-EGFR activatableantibody includes a masking moiety (MM), a cleavable moiety (CM) that iscleaved by the cleaving agent, and an antigen binding domain or fragmentthereof (AB) that specifically binds EGFR, wherein the anti-EGFRactivatable antibody in an uncleaved (i.e., non-activated) statecomprises a structural arrangement from N-terminus to C-terminus asfollows: MM-CM-AB or AB-CM-MM; (ii) wherein the MM is a peptide thatinhibits binding of the AB to EGFR, and wherein the MM does not have anamino acid sequence of a naturally occurring binding partner of the AB;and (iii) wherein the MM of the activatable antibody in an uncleavedstate interferes with specific binding of the AB to EGFR, and whereinthe MM of an activatable antibody in a cleaved (i.e., activated) statedoes not interfere or compete with specific binding of the AB to EGFR.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an anti-EGFR activatable antibody, wherein the anti-EGFRactivatable antibody includes a detectable label that is positioned on aportion of the anti-EGFR activatable antibody that is released followingcleavage of the CM; and (ii) measuring a level of detectable label inthe subject or sample, wherein a detectable level of the detectablelabel in the subject or sample indicates that the cleaving agent isabsent in the subject or sample and wherein no detectable level of thedetectable label in the subject or sample indicates that the cleavingagent is present in the subject or sample. Such an anti-EGFR activatableantibody includes a masking moiety (MM), a cleavable moiety (CM) that iscleaved by the cleaving agent, and an antigen binding domain or fragmentthereof (AB) that specifically binds EGFR, wherein the anti-EGFRactivatable antibody in an uncleaved (i.e., non-activated) statecomprises a structural arrangement from N-terminus to C-terminus asfollows: MM-CM-AB or AB-CM-MM; (ii) wherein the MM is a peptide thatinhibits binding of the AB to EGFR, and wherein the MM does not have anamino acid sequence of a naturally occurring binding partner of the AB;and (iii) wherein the MM of the activatable antibody in an uncleavedstate interferes with specific binding of the AB to EGFR, and whereinthe MM of an activatable antibody in a cleaved (i.e., activated) statedoes not interfere or compete with specific binding of the AB to EGFR.

The invention also provides kits for use in methods of detectingpresence or absence of a cleaving agent and a target of interest (EGFR)in a subject or a sample, where the kits include at least an anti-EGFRactivatable antibody and/or conjugated anti-EGFR activatable antibodydescribed herein for use in contacting a subject or sample and means fordetecting the level of activated anti-EGFR activatable antibody and/orconjugated anti-EGFR activatable antibody in the subject or sample,wherein a detectable level of the detectable label in the subject orsample indicates that the cleaving agent is absent in the subject orsample and wherein no detectable level of the detectable label in thesubject or sample indicates that the cleaving agent is present in thesubject or sample.

In some embodiments of these methods and/or kits, the anti-EGFRactivatable antibody includes a detectable label. In some embodiments ofthese methods and/or kits, the detectable label includes an imagingagent, a contrasting agent, an enzyme, a fluorescent label, achromophore, a dye, one or more metal ions, or a ligand-based label. Insome embodiments of these methods and/or kits, the imaging agentcomprises a radioisotope. In some embodiments of these methods, theradioisotope is indium or technetium. In some embodiments of thesemethods, the radioisotope is or is derived from iodine. In someembodiments of these methods, the radioisotope is ¹²⁵I or ¹³³I. In someembodiments of these methods and/or kits, the contrasting agentcomprises iodine, gadolinium or iron oxide. In some embodiments of thesemethods and/or kits, the enzyme comprises horseradish peroxidase,alkaline phosphatase, or β-galactosidase. In some embodiments of thesemethods and/or kits, the fluorescent label comprises yellow fluorescentprotein (YFP), cyan fluorescent protein (CFP), green fluorescent protein(GFP), modified red fluorescent protein (mRFP), red fluorescent proteintdimer2 (RFP tdimer2), HCRED, or a europium derivative. In someembodiments of these methods and/or kits, the luminescent labelcomprises an N-methylacrydium derivative. In some embodiments of thesemethods and/or kits, the label comprises an Alexa Fluor® label, such asAlex Fluor® 680 or Alexa Fluor® 750. In some embodiments of thesemethods and/or kits, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods and/or kits, the subject is amammal. In some embodiments of these methods and/or kits, the subject isa human. In some embodiments, the subject is a non-human mammal, such asa non-human primate, companion animal (e.g., cat, dog, horse), farmanimal, work animal, or zoo animal.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods and/or kits, the method and/or kit isused to identify or otherwise refine, e.g., stratify, a patientpopulation suitable for treatment with an anti-EGFR activatable antibodyof the disclosure. For example, patients that test positive for both thetarget (e.g., EGFR) and a protease that cleaves the substrate in thecleavable moiety (CM) of the anti-EGFR activatable antibody being testedin these methods are identified as suitable candidates for treatmentwith such an anti-EGFR activatable antibody comprising such a CM.Likewise, patients that test negative for either or both of the target(e.g., EGFR) and the protease that cleaves the substrate in the CM inthe activatable antibody being tested using these methods are identifiedas suitable candidates for another form of therapy (i.e., not suitablefor treatment with the anti-EGFR activatable antibody being tested). Insome embodiments, such patients can be tested with other anti-EGFRactivatable antibodies until a suitable anti-EGFR activatable antibodyfor treatment is identified (e.g., an anti-EGFR activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

The invention also provides kits for use in methods of identifying orotherwise refining a patient population, where the kits include at least(i) an anti-EGFR activatable antibody and/or conjugated anti-EGFRactivatable antibody described herein for use in contacting a subject orsample, (ii) means for detecting the level of activated anti-EGFRactivatable antibody and/or conjugated anti-EGFR activatable antibody inthe subject or sample, wherein a detectable level of activated anti-EGFRactivatable antibody in the sample indicates that the sample is positivefor the presence of EGFR and a cleaving agent that cleaves the substratein the cleavable moiety (CM) of the anti-EGFR activatable antibodyand/or conjugated anti-EGFR activatable antibody and (iii) means foridentifying and selecting one or more subjects that test positive forthe presence of EGFR and the cleaving agent thereby identifying orrefining a patient population. In some embodiments, the kit alsoincludes instructions for administering a therapeutically effectiveamount of an anti-EGFR activatable antibody and/or conjugated anti-EGFRactivatable antibody described herein to the one or more subjects in thepatient population that test positive for the presence of EGFR and thecleaving agent. In some embodiments, the kit also includes instructionsfor administering a therapeutically effective amount of anotheranti-EGFR therapeutic agent described herein to the one or more subjectsin the patient population that did not test positive for the presence ofboth EGFR and the cleaving agent. In some embodiments, the anti-EGFRactivatable antibody comprises a detectable label. In some embodiments,the detectable label comprises an imaging agent, a contrasting agent, anenzyme, a fluorescent label, a chromophore, a dye, one or more metalions, or a ligand-based label. In some embodiments, the EGFR-relateddisorder is cancer. In some embodiments, the cancer is breast cancer,colorectal cancer, gastric cancer, glioblastoma, head and neck cancer,lung cancer, ovarian cancer, endometrial cancer, pancreatic cancer,prostate cancer, renal cancer, sarcoma, or skin cancer. In someembodiments, the EGFR-related disorder is psoriasis.

In some embodiments of these methods, the MM is a peptide having alength from about 2 to 40 amino acids. In some embodiments of thesemethods, the anti-EGFR activatable antibody comprises a linker peptide,wherein the linker peptide is positioned between the MM and the CM. Insome embodiments of these methods, the anti-EGFR activatable antibodycomprises a linker peptide, where the linker peptide is positionedbetween the AB and the CM. In some embodiments of these methods, theanti-EGFR activatable antibody comprises a first linker peptide (L1) anda second linker peptide (L2), wherein the first linker peptide ispositioned between the MM and the CM and the second linker peptide ispositioned between the AB and the CM. In some embodiments of thesemethods, each of L1 and L2 is a peptide of about 1 to 20 amino acids inlength, and wherein each of L1 and L2 need not be the same linker. Insome embodiments of these methods, one or both of L1 and L2 comprises aglycine-serine polymer.

In some embodiments of these methods, at least one of LP1 or LP2comprises an amino acid sequence selected from the group consisting of(GS)_(n), (GGS)_(n), (GSGGS)_(n) (SEQ ID NO: 15) and (GGGS)_(n) (SEQ IDNO: 16), where n is an integer of at least one. In some embodiments ofthese methods, at least one of LP1 or LP2 comprises an amino acidsequence selected from the group consisting of GGSG (SEQ ID NO: 17),GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19), GSGGG (SEQ ID NO: 20),GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22). In some embodiments ofthese methods, LP1 comprises the amino acid sequence GSSGGSGGSGGSG (SEQID NO: 23). In some embodiments of these methods, LP2 comprises theamino acid sequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments of these methods, the AB comprises an antibody orantibody fragment sequence selected from the anti-EGFR antibodysequences presented herein. In some embodiments of these methods, the ABcomprises a Fab fragment, a scFv or a single chain antibody (SCAB).

In some embodiments of these methods, the cleaving agent is a proteasethat is co-localized in the subject or sample with EGFR and the CM is apolypeptide that functions as a substrate for the protease, wherein theprotease cleaves the CM in the anti-EGFR activatable antibody when theanti-EGFR activatable antibody is exposed to the protease. In someembodiments of these methods, the CM is a polypeptide of up to 15 aminoacids in length. In some embodiments of these methods, the CM is coupledto the N-terminus of the AB. In some embodiments of these methods, theCM is coupled to the C-terminus of the AB. In some embodiments of thesemethods, the CM is coupled to the N-terminus of a VL chain of the AB.

In some embodiments of these methods, the cleaving agent is an enzymeand the CM is a substrate for the enzyme. In some embodiments of thesemethods, the enzyme is a protease disclosed herein. In some embodimentsof these methods, the protease is one of the proteases disclosed herein.In some embodiments of these methods, the protease is selected from thegroup consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3,TMPRSS4, MMP-9, MMP-12, MMP-13, and MMP-14. In some embodiments, theenzyme comprises uPA. In some embodiments, the enzyme compriseslegumain. In some embodiments, the enzyme comprises MT-SP1. In someembodiments, the protease is not active or is significantly less activein tissues that do not significantly express EGFR. In some embodiments,the protease is not active or is significantly less active in healthy,e.g., non-diseased tissues.

The invention also provides methods of using the anti-EGFR antibodiesand/or conjugated activatable anti-EGFR antibodies (i.e., activatableanti-EGFR antibody conjugates, also referred to herein as conjugatedactivatable anti-EGFR antibodies and/or conjugated anti-EGFR activatableantibodies) in a variety of diagnostic and/or prophylactic indications.For example, the invention provides methods of detecting presence orabsence of a cleaving agent and a target of interest (EGFR) in a subjector a sample by (i) contacting a subject or sample with an anti-EGFRantibody and/or conjugated activatable anti-EGFR antibody and (ii)measuring a level of anti-EGFR antibody and/or conjugated activatableanti-EGFR antibody in the subject or sample, wherein a detectable levelof activated anti-EGFR antibody and/or conjugated activatable anti-EGFRantibody in the subject or sample indicates that the cleaving agent andEGFR are present in the subject or sample and wherein no detectablelevel of activated anti-EGFR antibody and/or conjugated activatableanti-EGFR antibody in the subject or sample indicates that the cleavingagent, EGFR or both the cleaving agent and EGFR are absent in thesubject or sample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an anti-EGFR antibody and/or conjugated activatableanti-EGFR antibody in the presence of EGFR, and (ii) measuring a levelof activated anti-EGFR antibody and/or conjugated activatable anti-EGFRantibody in the subject or sample, wherein a detectable level ofactivated anti-EGFR antibody and/or conjugated activatable anti-EGFRantibody in the subject or sample indicates that the cleaving agent ispresent in the subject or sample and wherein no detectable level ofanti-EGFR antibody and/or conjugated activatable anti-EGFR antibody inthe subject or sample indicates that the cleaving agent is absent in thesubject or sample.

The invention also provides methods of detecting presence or absence ofa cleaving agent in a subject or a sample by (i) contacting a subject orsample with an anti-EGFR antibody and/or conjugated activatableanti-EGFR antibody; and (ii) measuring a level of detectable label inthe subject or sample, wherein a detectable level of the detectablelabel in the subject or sample indicates that the cleaving agent isabsent in the subject or sample and wherein no detectable level of thedetectable label in the subject or sample indicates that the cleavingagent is present in the subject or sample.

In some embodiments of these methods, the anti-EGFR antibody and/orconjugated activatable anti-EGFR antibody includes a detectable labelselected from the group consisting of an imaging agent, a contrastingagent, an enzyme, a fluorescent label, a chromophore, a dye, one or moremetal ions, and a ligand-based label. In some embodiments of thesemethods, the imaging agent comprises a radioisotope. In some embodimentsof these methods, the radioisotope is indium or technetium. In someembodiments of these methods, the contrasting agent comprises iodine,gadolinium or iron oxide. In some embodiments of these methods, theenzyme comprises horseradish peroxidase, alkaline phosphatase, orβ-galactosidase. In some embodiments of these methods, the fluorescentlabel comprises yellow fluorescent protein (YFP), cyan fluorescentprotein (CFP), green fluorescent protein (GFP), modified red fluorescentprotein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, ora europium derivative. In some embodiments of these methods, theluminescent label comprises an N-methylacrydium derivative. In someembodiments of these methods, the label comprises an Alexa Fluor® label,such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments ofthese methods, the ligand-based label comprises biotin, avidin,streptavidin or one or more haptens.

In some embodiments of these methods, the subject is a mammal. In someembodiments of these methods, the subject is a human. In someembodiments, the subject is a non-human mammal, such as a non-humanprimate, companion animal (e.g., cat, dog, horse), farm animal, workanimal, or zoo animal.

In some embodiments of these methods, the method is an in vivo method.In some embodiments of these methods, the method is an in situ method.In some embodiments of these methods, the method is an ex vivo method.In some embodiments of these methods, the method is an in vitro method.

In some embodiments of the methods, the method is used to identify orotherwise refine a patient population suitable for treatment with ananti-EGFR antibody and/or conjugated activatable anti-EGFR antibody ofthe disclosure. For example, patients that test positive for both thetarget (e.g., EGFR) and a protease that cleaves the substrate in thecleavable moiety (CM) of the anti-EGFR antibody and/or conjugatedactivatable anti-EGFR antibody being tested in these methods areidentified as suitable candidates for treatment with such anti-EGFRantibody and/or such a conjugated activatable anti-EGFR antibodycomprising such a CM. Likewise, patients that test negative for eitheror both of the target (e.g., EGFR) and the protease that cleaves thesubstrate in the CM in the activatable antibody being tested using thesemethods are identified as suitable candidates for another form oftherapy (i.e., not suitable for treatment with the anti-EGFR antibodyand/or conjugated activatable anti-EGFR antibody being tested). In someembodiments, such patients can be tested with other anti-EGFR antibodyand/or conjugated activatable anti-EGFR antibody until a suitableanti-EGFR antibody and/or conjugated activatable anti-EGFR antibody fortreatment is identified (e.g., an anti-EGFR antibody and/or conjugatedactivatable anti-EGFR antibody comprising a CM that is cleaved by thepatient at the site of disease).

In some embodiments, the activatable antibody binds EGFR in an activatedstate and includes (i) an antibody or an antigen binding fragmentthereof (AB) that specifically binds to EGFR, wherein the AB is or isderived from cetuximab; (ii) a masking moiety (MM) that inhibits thebinding of the AB of the activatable antibody in an uncleaved state toEGFR; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CMis a polypeptide that functions as a substrate for a protease.

In some embodiments, the MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to EGFR. In some embodiments, the MM has anequilibrium dissociation constant for binding to the AB which is no morethan the equilibrium dissociation constant of the AB to EGFR. In someembodiments, the MM does not interfere or compete with the AB of theactivatable antibody in a cleaved state for binding to EGFR when theactivatable antibody is.

In some embodiments, the protease is co-localized with EGFR in a tissue,and the protease cleaves the CM in the activatable antibody when theactivatable antibody is exposed to the protease. In some embodiments,the protease is not active or is significantly less active in tissuesthat do not significantly express EGFR. In some embodiments, theprotease is not active or is significantly less active in healthy, e.g.,non-diseased tissues.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody has the structural arrangement from N-terminus toC-terminus as follows in the uncleaved state: MM-CM-AB or AB-CM-MM.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody includes a linking peptide between the MM and theCM.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody includes a linking peptide between the CM and theAB.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody includes a first linking peptide (LP1) and a secondlinking peptide (LP2), and wherein the activatable antibody and/orconjugated activatable antibody has the structural arrangement fromN-terminus to C-terminus as follows in the uncleaved state:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the twolinking peptides need not be identical to each other.

In some embodiments, the AB has an equilibrium dissociation constant ofabout 100 nM or less for binding to EGFR.

In some embodiments, the MM is a polypeptide of about 2 to 40 aminoacids in length, for example, no more than 40 amino acids long.

In some embodiments, the MM polypeptide sequence is different from thatof EGFR, and the MM polypeptide sequence is no more than 50% identicalto any natural binding partner of the AB.

In some embodiments, the CM is positioned in the activatable antibodyand/or conjugated activatable antibody such that in the uncleaved state,binding of the activatable antibody and/or conjugated activatableantibody to EGFR is reduced to occur with an equilibrium dissociationconstant that is at least 20-fold greater than the equilibriumdissociation constant of an unmodified AB binding to EGFR, and whereasthe AB of the activatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodyand/or conjugated activatable antibody such that in the uncleaved state,binding of the activatable antibody and/or conjugated activatableantibody to EGFR is reduced to occur with an equilibrium dissociationconstant that is at least 100-fold greater than the equilibriumdissociation constant of an unmodified AB binding to EGFR, and whereasthe AB of the activatable antibody in the cleaved state binds EGFR.

In some embodiments, the CM is positioned in the activatable antibodyand/or conjugated activatable antibody such that in the uncleaved state,binding of the activatable antibody and/or conjugated activatableantibody to EGFR is reduced to occur with an equilibrium dissociationconstant that is at least 200-fold greater than the equilibriumdissociation constant of an unmodified AB binding to EGFR, and whereasthe AB of the activatable antibody in the cleaved state binds EGFR.

In some embodiments, the coupling of the MM reduces the ability of theAB to bind EGFR such that the dissociation constant (K_(d)) of the ABwhen coupled to the MM towards EGFR is at least 20 times greater thanthe K_(d) of the AB when not coupled to the MM towards EGFR. In someembodiments, the coupling of the MM reduces the ability of the AB tobind EGFR such that the K_(d) of the AB when coupled to the MM towardsEGFR is at least 100 times greater than the K_(d) of the AB when notcoupled to the MM towards EGFR. In some embodiments, the coupling of theMM reduces the ability of the AB to bind EGFR such that the K_(d) of theAB when coupled to the MM towards EGFR is at least 1000 times greaterthan the K_(d) of the AB when not coupled to the MM towards EGFR. Insome embodiments, the coupling of the MM reduces the ability of the ABto bind EGFR such that the K_(d) of the AB when coupled to the MMtowards EGFR is at least 10,000 times greater than the K_(d) of the ABwhen not coupled to the MM towards EGFR.

In some embodiments, the CM is a polypeptide of up to 15 amino acids inlength.

In some embodiments, in the presence of EGFR, the MM reduces the abilityof the AB to bind EGFR by at least 90% when the CM is uncleaved, ascompared to when the CM is cleaved when assayed in vitro using a targetdisplacement assay such as, for example, the assay described in PCTPublication Nos. WO 2009/025846 and WO 2010/081173.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody includes an antibody or antigen-binding fragmentthereof that specifically binds EGFR. In some embodiments, the antibodyor immunologically active fragment thereof that binds EGFR is amonoclonal antibody, domain antibody, single chain, Fab fragment, aF(ab′)₂ fragment, a scFv, a scab, a dAb, a single domain heavy chainantibody, and a single domain light chain antibody. In some embodiments,such an antibody or immunologically active fragment thereof that bindsEGFR is a mouse, chimeric, humanized or fully human monoclonal antibody.

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:6, and SEQ ID NO: 10, and a light chain amino acid sequence includingSEQ ID NO: 68. In some embodiments, the AB of the activatable antibodyand/or conjugated activatable antibody includes a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid selected from the group consisting of SEQID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an amino acid sequence including SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody is encoded by a nucleic acid sequencethat includes a nucleic acid sequence encoding a heavy chain amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:6, and SEQ ID NO: 10, and a nucleic acid encoding a light chain aminoacid sequence including SEQ ID NO: 68. In some embodiments, the AB ofthe activatable antibody and/or conjugated activatable antibody isencoded by a nucleic acid sequence encoding a includes a heavy chainamino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to an amino acid selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, and anucleic acid sequence that includes a nucleic acid sequence encoding alight chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence including SEQID NO: 68. In some embodiments, the activatable antibody includes thelight chain amino acid sequence of SEQ ID NO: 68 and a spacer amino acidsequence. In some embodiments, the activatable antibody includes thelight chain amino acid of SEQ ID NO: 68 and a spacer amino acid sequencethat includes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid sequence of SEQ ID NO: 68 joineddirectly to a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid of SEQ ID NO: 68 and a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid sequence of SEQ ID NO: 68 joineddirectly to a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody is encoded by a nucleic acid including aheavy chain nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and a light chain nucleicacid sequence of SEQ ID NO: 67. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody is encodedby a nucleic acid sequence including a heavy chain nucleic acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to an nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, and a nucleic acidsequence that includes a nucleic acid sequence encoding a light chainnucleic acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to a nucleic acid sequence including SEQ ID NO: 67.

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30 and SEQ ID NO: 34, and a light chain amino acid sequence includingSEQ ID NO: 68. In some embodiments, the AB of the activatable antibodyand/or conjugated activatable antibody includes a heavy chain amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30 and SEQ ID NO: 34, and alight chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence sincluding SEQ ID NO: 68. In some embodiments, the activatable antibodyincludes the light chain amino acid sequence of SEQ ID NO: 68 and aspacer amino acid sequence. In some embodiments, the activatableantibody includes the light chain amino acid of SEQ ID NO: 68 and aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid sequence ofSEQ ID NO: 68 joined directly to a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid of SEQ ID NO: 68 and a spaceramino acid sequence that includes the amino acid sequence QGQSGQ (SEQ IDNO: 38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid sequence of SEQID NO: 68 joined directly to a spacer amino acid sequence that includesthe amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody is encoded by a nucleic acid sequenceencoding a heavy chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and anucleic acid sequence encoding a light chain amino acid sequenceincluding SEQ ID NO: 68. In some embodiments, the AB of the activatableantibody and/or conjugated activatable antibody is encoded by a nucleicacid sequence that includes a nucleic acid sequence encoding a includesa heavy chain amino acid sequence that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid including SEQID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, and a nucleic acid sequenceencoding a light chain amino acid that is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceincluding SEQ ID NO: 68. In some embodiments, the activatable antibodyincludes the light chain amino acid sequence of SEQ ID NO: 68 and aspacer amino acid sequence. In some embodiments, the activatableantibody includes the light chain amino acid of SEQ ID NO: 68 and aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid sequence ofSEQ ID NO: 68 joined directly to a spacer amino acid sequence thatincludes the amino acid sequence QGQSGQ (SEQ ID NO: 38). In someembodiments, the activatable antibody includes a light chain amino acidthat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the light chain amino acid of SEQ ID NO: 68 and a spaceramino acid sequence that includes the amino acid sequence QGQSGQ (SEQ IDNO: 38). In some embodiments, the activatable antibody includes a lightchain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical to the light chain amino acid sequence of SEQID NO: 68 joined directly to a spacer amino acid sequence that includesthe amino acid sequence QGQSGQ (SEQ ID NO: 38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody is encoded by a nucleic acid including aheavy chain nucleic acid sequence selected from the group consisting ofSEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, and a light chainnucleic acid sequence including SEQ ID NO: 67. In some embodiments, theAB of the activatable antibody and/or conjugated activatable antibody isencoded by a nucleic acid sequence including a heavy chain nucleic acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to an nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 25, SEQ ID NO: 29, and SEQ ID NO: 33, and anucleic acid sequence encoding a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to anucleic acid sequence including SEQ ID NO: 67.

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 2, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 2, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 26, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 26, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 6, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 6, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 30, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 30, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 10, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 10, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the AB of the activatable antibody and/orconjugated activatable antibody includes a heavy chain including theamino acid sequence of SEQ ID NO: 34, and a light chain including theamino acid sequence of SEQ ID NO: 68. In some embodiments, the AB of theactivatable antibody and/or conjugated activatable antibody includes aheavy chain including an amino acid sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acidsequence of SEQ ID NO: 34, and a light chain including an amino acidsequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the amino acid sequence of SEQ ID NO: 68. In someembodiments, the activatable antibody includes the light chain aminoacid sequence of SEQ ID NO: 68 and a spacer amino acid sequence. In someembodiments, the activatable antibody includes the light chain aminoacid of SEQ ID NO: 68 and a spacer amino acid sequence that includes theamino acid sequence QGQSGQ (SEQ ID NO: 38). In some embodiments, theactivatable antibody includes a light chain amino acid that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thelight chain amino acid sequence of SEQ ID NO: 68 joined directly to aspacer amino acid sequence that includes the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, the activatable antibody includesa light chain amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the light chain amino acid of SEQ IDNO: 68 and a spacer amino acid sequence that includes the amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, the activatableantibody includes a light chain amino acid that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the light chainamino acid sequence of SEQ ID NO: 68 joined directly to a spacer aminoacid sequence that includes the amino acid sequence QGQSGQ (SEQ ID NO:38).

In some embodiments, the CM is a substrate for an enzyme selected fromthe group consisting of uPA, legumain and MT-SP1. In some embodiments,the enzyme comprises uPA. In some embodiments, the enzyme compriseslegumain. In some embodiments, the enzyme comprises MT-SP1.

In some embodiments, the CM includes the amino acid sequence LSGRSDNH(SEQ ID NO: 13).

In some embodiments, the MM does not include more than 25% amino acidsequence identity to EGFR. In some embodiments, the MM does not includemore than 10% amino acid sequence identity to EGFR.

In some embodiments, the MM includes the amino acid sequenceCISPRGCPDGPYVMY (SEQ ID NO: 14).

In some embodiments, at least one of LP1 or LP2 includes an amino acidsequence selected from the group consisting of (GS)_(n), (GGS)_(n),(GSGGS)_(n) (SEQ ID NO: 15) and (GGGS)_(n) (SEQ ID NO: 16), where n isan integer of at least one. In some embodiments, at least one of LP1 orLP2 includes an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19),GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), and GSSSG (SEQ ID NO: 22).In some embodiments, LP1 includes the amino acid sequence GSSGGSGGSGGSG(SEQ ID NO: 23). In some embodiments, LP2 includes the amino acidsequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).

In some embodiments, the activatable antibody also includes an agentconjugated to the AB. In some embodiments, the agent is a therapeuticagent. In some embodiments, the agent is an antineoplastic agent. Insome embodiments, the agent is a toxin or fragment thereof. In someembodiments, the agent is an agent selected from the group listed inTable 1. In some embodiments, the agent is a dolastatin. In someembodiments, the agent is an auristatin or derivative thereof. In someembodiments, the agent is auristatin E or a derivative thereof. In someembodiments, the agent is monomethyl auristatin E (MMAE). In someembodiments, the agent is a maytansinoid or maytansinoid derivative. Insome embodiments, the agent is DM1 or DM4. In some embodiments, theagent is a duocarmycin or derivative thereof. In some embodiments, theagent is a calicheamicin or derivative thereof.

In some embodiments, the agent is conjugated to the AB via a linker. Insome embodiments, the linker is a cleavable linker. In some embodiments,the linker is selected from the group consisting of the linkers shown inTables 2 and 3.

In some embodiments, the activatable antibody and/or conjugatedactivatable antibody also includes a signal peptide. In someembodiments, the signal peptide is conjugated to the activatableantibody and/or conjugated activatable antibody via a spacer. In someembodiments, the spacer is conjugated to the activatable antibody and/orconjugated activatable antibody in the absence of a signal peptide. Insome embodiments, the spacer is joined directly to the MM of theactivatable antibody and/or conjugated activatable antibody. In someembodiments, the spacer includes at least the amino acid sequence QGQSGQ(SEQ ID NO: 38). In some embodiments, an activatable antibody and/orconjugated activatable antibody includes a spacer of sequence QGQSGQ(SEQ ID NO: 38) joined directly to a MM sequence CISPRGCPDGPYVMY (SEQ IDNO: 14) in the structural arrangement from N-terminus to C-terminus ofspacer-MM-CM-AB. In some embodiments, an activatable antibody and/orconjugated activatable antibody includes a spacer joined directly to aMM sequence and includes the amino acid sequence QGQSGQCISPRGCPDGPYVMY(SEQ ID NO: 59) in the structural arrangement from N-terminus toC-terminus of spacer-MM-CM-AB.

In some embodiments, the activatable anti-EGFR antibody and/orconjugated activatable anti-EGFR antibody is monospecific. In someembodiments, the activatable anti-EGFR antibody and/or conjugatedactivatable anti-EGFR antibody is multispecific, e.g., by way ofnon-limiting example, bispecific or trifunctional. In some embodiments,the activatable anti-EGFR antibody and/or conjugated activatableanti-EGFR antibody is formulated as part of a pro-Bispecific T CellEngager (BITE) molecule. In some embodiments, the activatable anti-EGFRantibody and/or conjugated activatable anti-EGFR antibody is formulatedas part of a pro-Chimeric Antigen Receptor (CAR) modified T cell orother engineered receptor.

Pharmaceutical compositions according to the invention can include anantibody and/or conjugated antibody of the invention and a carrier.These pharmaceutical compositions can be included in kits, such as, forexample, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration depicting an activatable antibody of theinvention being masked in a first state and activated in the presence ofa disease-associated protease.

FIG. 1B is an illustration depicting the substrate selection processused to identify suitable substrates that will selectively activate theactivatable anti-EGFR antibodies of the invention at the desiredlocation.

FIG. 2A is a schematic representation of an activatable antibody.

FIGS. 2B-2D are a series of graphs depicting the in vitrocharacterization of an anti-EGFR activatable antibody referred to hereinas “Pb-1204” or 3954-1204-C225v4 activatable antibody. FIG. 2B is agraph depicting the enzymatic efficiency of two proteases, MT-SP1 anduPA, at cleaving Pb-1204. FIG. 2C is a graph depicting capillaryelectrophoretic analysis of Pb-1204 before and after digestion with theproteases uPA, MT-SP1 and legumain.

FIG. 2D is a graph depicting the effect of the anti-EGFR antibodycetuximab, Pb-1204 and uPA-activated Pb-1204 on the proliferation ofH292 cells.

FIG. 3A is a schematic representation of the chemical structure of thenear-infrared quenched probe Cy5.5-1204-Q.

FIGS. 3B-3H are a series of illustrations depicting how the activatableanti-EGFR antibody referred to herein as Pb-1204 accumulates inxenograft tumors. FIG. 3B is an illustration depicting the opticalimaging of HT29 xenograft tumor bearing mice injected intraperitoneallywith either of Alexa Fluor 750-conjugated Pb-NSUB (red) and quenchedprobe Cy5-NSUB-Q, referred to herein as “IQ-NSUB” (blue; left panel),where NSUB represents an amino acid sequence that is not susceptible toprotease cleavage, or Alexa Fluor 750-conjugated Pb-1204 (red) andquenched probe Cy5.5-1204-Q, referred to herein as “IQ-1204” (blue;right panel), where 1204 is the substrate sequence 1204: LSGRSDNH (SEQID NO: 13), which is susceptible to cleavage by at least uPA. Ahigh-intensity overlapping fluorescent signal was detected only in thetumor of mouse administered with activatable anti-EGFR antibody andprobe containing 1204 substrate. FIGS. 3C and 3D are illustrationsdepicting immunofluorescent detection of activatable anti-EGFRantibodies in HT29 xenograft tumors 7 days after the mice were injectedwith Alexa Fluor 750-conjugated Pb-1204 (FIG. 3C) or Alexa Fluor750-conjugated Pb-NSUB (FIG. 3D). FIGS. 3E and 3F are a series ofphotographs the optical imaging of H292 xenograft tumor bearing miceinjected intraperitoneally with Pb-NSUB, 3954-1204-C225v4 or cetuximab(i.e., unmodified cetuximab). The 3954-1204-C225v4 activatable antibodylocalizes to the tumor site in the H292 xenograft mouse model. FIG. 3Gis a series of photographs that depict the three dimensional optical andcomputed tomography (3D/CT tomography) of animals in the H292 xenograftmouse model that were administered cetuximab, Pb-NSUB or3954-1204-C225v4. FIG. 3H is a series of photographs depicting human IgGimmunohistochemistry staining on representative H292 tumors from in vivoimaging studies.

FIGS. 4A-4F are a series of graphs depicting the ability of theactivatable anti-EGFR antibody referred to herein as Pb-1204 to inhibittumor growth in xenograft models. FIG. 4A is a graph depicting theeffects seen in H292 xenograft tumor-bearing mice that were treatedweekly using cetuximab (25 mg/kg, blue line), Pb-1204 (25 mg/kg, redline), Pb-NSUB (25 mg/kg, green line), or vehicle (black line), wherePb-NSUB represents an activatable anti-EGFR antibody construct that hasan antibody portion that binds EGFR and also includes the NSUB aminoacid sequence, which is not susceptible to protease cleavage. Data arepresented as mean tumor volume±SEM. Percent tumor growth inhibition(TGI) was evaluated at Day 20. FIGS. 4B and 4C are graphs depicting thelevel of IgG concentration and the level of EGFR-binding IgG in samplesof plasma (1, 8, 24, 72 h post-dose) (FIG. 4B) and tumor (72 hpost-dose) (FIG. 4C) collected from H292 xenograft tumor-bearing miceinjected with 25 mg/kg of cetuximab, Pb-NSUB or Pb-1204. IgGconcentration (blue) and EGFR binding (red) were measured by ELISA. FIG.4D is a graph depicting the tumor volume observed in LXFA677tumor-bearing mice that were injected intraperitoneally (IP) once withcetuximab (25 mg/kg, blue line), Pb-1204 (25 mg/kg, red line), vehicle(black line). Data are presented as mean tumor volume±SEM. Percent tumorgrowth inhibition (TGI) was evaluated at Day 25. FIGS. 4E and 4F aregraphs depicting the level of IgG concentration and the level ofEGFR-binding IgG in samples of plasma (day 1, 7, 14, 21, 28 post-dose)(FIG. 4E) and tumor (72 h post-dose) (FIG. 4F) collected from LXFA677xenograft tumor-bearing mice injected with vehicle, 25 mg/kg ofcetuximab or 25 mg/kg of Pb-1204. The IgG concentration (blue) and theEGFR binding (red) were measured by ELISA.

FIGS. 5A-5C are a series of graphs showing further in vitrocharacterization of activatable anti-EGFR antibodies. FIG. 5A is a graphdepicting the level of proteolytic conversion of Pb-1204 cleavage byMT-SP1 and uPA. FIG. 5B is a photograph of SDS-PAGE analysis of Pb-1204before and after digestion with legumain, MT-SP1 and uPA. FIG. 5C is agraph depicting the binding of cetuximab, parental Ab, Pb-1204 oruPA-activated Pb-1204 to immobilized EGFR-ECD.

FIGS. 6A and 6B are illustrations depicting how Pb-1204 is not activatedin normal liver. FIGS. 6A and 6B depict the immunofluorescent detectionof activatable anti-EGFR antibodies in liver 7 days after the mice wereinjected with Alexa Fluor 750-conjugated Pb-1204 (FIG. 6A) or AlexaFluor 750-conjugated Pb-NSUB (FIG. 6B).

FIGS. 7A and 7B are an illustration and a graph depicting tumoractivation and PK of cetuximab and Pb-1204 in tumor bearing mice. InFIG. 7A, lysates of cetuximab, Pb-NSUB and Pb-1204 (25 mg/kg) treatedH292 xenograft tumors were generated 72 h post-treatment and human IgGlight chain was analyzed by immunoblotting. In FIG. 7B, plasma fromLXFA677 xenograft tumor-bearing mice injected on day 0 with 25 mg/kg ofcetuximab or Pb-1204 was collected at days 1, 7, 14, 21, 28 post-dose,and the binding to immobilized EGFR-ECD (cetuximab, blue; Pb-1204, red)was measured.

FIGS. 8A and 8B are a series of illustrations depicting the progression,invasion and metastasis of tumors that result from severalinterdependent processes in which proteases are implicated. Thesefigures are adapted from Affara N I, et al. “Delineating proteasefunctions during cancer development.” Methods Mol Biol. 539 (2009):1-32.

FIGS. 9A and 9B are a table and graph that depict the rate of cleavageand substrate selectivity of the proteases uPA (human and mouse), MT-SP1(human and mouse), legumain (human), tissue plasminogen activator (tPA,human) and thrombin (human) for the 1204 substrate sequence LSGRSDNH(SEQ ID NO: 13) in an IQ probe. FIGS. 9C and 9D are a series of graphsthat depict the rate of cleavage of the 1204 substrate sequence byrecombinant human uPA (FIG. 9C) and recombinant human MT-SP1 (FIG. 9D).The 1204 substrate sequence was present at a final concentration of 500nM in each study.

FIG. 10A is a graph depicting the relative ability of unmodifiedcetuximab antibody (“Parental”), the Pb-NSUB construct (“NSUB”), and theuncleaved 3954-1204-C225v4 activatable antibody to bind EGFR in theabsence of protease. FIG. 10B is a graph that depicts the relativeability of the unmodified cetuximab antibody (“Parental”), the Pb-NSUBconstruct (“NSUB”), and activated, i.e., cleaved 3954-1204-C225v4activatable antibody to bind EGFR in the presence of two proteases,human uPA and human MT-SP1. FIG. 10B demonstrates that once activated bythe appropriate protease, the 3954-1204-C225v4 activatable antibodybinds EGFR at comparable levels to the parental antibody.

FIGS. 11A and 11B are a series of graphs depicting that the activatableanti-EGFR antibody 3954-1204-C225v4, after at least four weeks afterdosing in mice, is stable in circulation and exhibits low activation inplasma, which suggests a superior toxicity profile. As used in FIGS. 11Aand 11B, “Parental Ab” refers to the unmodified cetuximab antibody, thePb-NSUB construct represents an activatable anti-EGFR antibody thatincludes the NSUB sequence that is not susceptible to protease cleavage,and “PB-NSELECT” is an activatable anti-EGFR antibody that includes anon-selective substrate, i.e., an amino acid sequence that is recognizedby enzymes in the circulation.

FIG. 12 is a graph depicting the number of non-human primates displayinga skin rash following administration with cetuximab, Pb-NSUBv5 (i.e.,3954-NSUB-C225v5) or 3954-1204-C225v5.

FIGS. 13A-13D are a series of graphs comparing the level of cetuximaband 3954-1204-C225v5 detected in the blood of the cynomolgus monkeysthat received 25 mg/kg weekly dosing after the initial 40 mg/kg loadingdose.

FIGS. 14A-14C are a series of photographs of fluorescent imaging in H292xenograft tumors with an antibody that specifically recognizes theactive site of MT-SP1 (antibody A11).

FIG. 15 is a schematic overview of in situ imaging of an activatableantibody: 1. A tissue section is laid over the slide. 2. The slide iscovered with solution containing labeled activatable antibody andincubated. 3. After extensive washing, binding of activated antibody isvisualized.

FIG. 16 is a series of photographs showing the ability of anti-EGFRactivatable antibody 3954-1204-C225v5 to be activated and to bind frozenhuman cancer tissues using in situ imaging. The red fluorescent tissueimage in FIG. 16, panel A demonstrates binding of C225v5 antibodyactivated by tissue-derived proteolytic cleavage of the anti-EGFRactivatable antibody. The identical pattern of tissue staining wasdetected by exposing a commercially available anti-EGFR antibody to thetissue, as shown in FIG. 16, panel B. FIG. 16, panel C demonstrates thatthe fluorescent signal shown in panel A was inhibited by pre-treatmentof the tissue with a 1:100 dilution of broad spectrum inhibitor cocktailset III and 50 mM EDTA. Blue staining represents DAPI nuclear staining.

FIG. 17 is a series of tables depicting that 3954-1204-C225v5 isactivatable in a wide range of human tumor samples. Column 2 indicatesthe expression level of EGFR receptor, as detected by a commerciallyavailable anti-EGFR antibody, for the various human cancer tissuesamples. Column 3 indicates the amount of active matriptase (MT-SP1), asdetected by antibody A11, in the various human cancer tissue samples.Columns 4 and 5 represent an evaluation of in situ activation andbinding of the EGFR activatable antibody (col. 5) as compared tocetuximab (Cetux) tissue staining (col. 4). The IHC staining thatmeasures the amount of EGFR and A11 antibodies binding to the tissuesample was scored from 0 to 3+: 0, no staining; 1+ (i.e., “+”), weakstaining; 2+ (i.e., “++”), moderate staining; and 3+ (i.e., “+++”),strong staining. The in situ imaging staining scoring is based oncomparison with cetuximab antibody staining and defined as follows: 0,no staining; 1+ (i.e., “+”), weak staining as compared to parentalantibody; 2+ (i.e., “++”), moderate staining as compared to parentalantibody; and 3+ (i.e., “+++”), analogous staining to parental antibody.

FIG. 18 is a graph depicting the ability of the anti-EGFR activatableantibody-agent conjugate 3954-1204-C225v5-MMAE to inhibit tumor growth.

FIG. 19 is a graph depicting the half-life of the anti-EGFR activatableantibody 3954-1204-C225v5 as compared to the half-life of cetuximab.

FIGS. 20A and 20B are a series of graphs depicting that lysineconjugation of MMAE increases 3954-1204-C225v5 potency while maintainingmasking and activation potential. The conjugation of MMAE to3954-1204-C225v5 does not alter its binding to EGFR. The conjugation ofMMAE to 3954-1204-C225v5 increases its cell killing activity.

FIG. 21 is an illustration depicting the co-localization of EGFR and A11in human colorectal cancer liver metastasis tissue samples.

FIG. 22 is an illustration depicting the ability of human colorectalcancer liver metastasis tissues to activate and bind anti-EGFRactivatable antibodies.

FIG. 23 is a series of photographs showing the triple staining of insitu imaging, EGFR IHC and A11 IHC. The upper row of images demonstratesthe staining performed on a single tissue slice, demonstrating (left toright): EGFR expression, activity of matriptase (MT-SP1) and binding ofcetuximab under in situ imaging conditions. The lower row of imagesdemonstrates the staining performed on a single tissue slice,demonstrating (left to right): EGFR expression, activity of matriptase(MT-SP1) and in situ imaging of anti-EGFR activatable antibody3954-1204-C225v5. The right column of images in FIG. 23 compares bindingof cetuximab (upper image) and of anti-EGFR activatable antibodyactivated by tissue-derived proteolytic cleavage (lower image) under insitu imaging conditions. The identical pattern of tissue staining wasdetected by exposing a commercially available anti-EGFR antibody to thetissue, as shown in FIG. 23, left column of images. FIG. 23, middlecolumn of images, demonstrates co-localization of matriptase (MT-SP1)activity with EGFR expression.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides activatable monoclonal antibodies (mAbs)that specifically bind human epidermal growth factor receptor (EGFR),also known as EGF receptor, human EGF receptor-1 (HER1), erbB, erbB1,and species antigen 7 (SA-7). The activatable anti-EGFR antibodies, alsoreferred to herein as anti-EGFR activatable antibodies or EGFRactivatable antibodies, are used in methods of treating, preventing,delaying the progression of, ameliorating and/or alleviating a symptomof a disease or disorder associated with aberrant EGFR expression and/oractivity. For example, the activatable anti-EGFR antibodies are used inmethods of treating, preventing, delaying the progression of,ameliorating and/or alleviating a symptom of a cancer or otherneoplastic condition.

The activatable anti-EGFR antibodies include an antibody orantigen-binding fragment thereof that specifically binds epidermalgrowth factor receptor (EGFR) coupled to a masking moiety (MM), suchthat coupling of the MM reduces the ability of the antibody orantigen-binding fragment thereof to bind EGFR. In a preferredembodiment, the MM is coupled via a sequence that includes a substratefor a protease, for example, a protease that is co-localized with EGFRat a treatment site in a subject. Numerous studies have demonstrated thecorrelation of aberrant protease levels, e.g., uPA, legumain, MT-SP1,matrix metalloproteases (MMPs), in solid tumors. (See e.g., Murthy R V,et al. “Legumain expression in relation to clinicopathologic andbiological variables in colorectal cancer.” Clin Cancer Res. 11 (2005):2293-2299; Nielsen B S, et al. “Urokinase plasminogen activator islocalized in stromal cells in ductal breast cancer.” Lab Invest 81(2001): 1485-1501; Mook O R, et al. “In situ localization ofgelatinolytic activity in the extracellular matrix of metastases ofcolon cancer in rat liver using quenched fluorogenic DQ-gelatin.” JHistochem Cytochem. 51 (2003): 821-829).

The activatable anti-EGFR antibodies provided herein include a substratefor a protease, which is useful in leveraging the protease activity intumor cells for targeted antibody activation at the site of treatmentand/or diagnosis. A general overview of this process is shown in FIG.1A, and a general overview of the process by which a suitable substratefor a protease such as uPA, MT-SP1, Legumain, etc., is selected is shownin FIG. 1B. The substrate selection process is used to identifysubstrates that have a number of desirable characteristics. For example,the selected substrates are systemically stable (i.e., stable in thesystemic circulation of a subject), are generally not susceptible tocleavage by circulating proteases such as plasmin, thrombin, tissueplasminogen activator (tPA) or a kallikrein (KLK) such as KLK-5 and/orKLK-7, are non-toxic, are generally not susceptible to cleavage atpotential sites of toxicity such as the skin by proteases such as ADAM9, ADAM 10, ADAM 17 and/or kallikreins, such as KLK-5 and KLK-7, and areactive at an intended site of treatment and/or diagnosis. Preferably,the identified substrates are selected for proteases that areoverexpressed at an intended site of therapy and/or diagnosis but arenot typically expressed at or in normal, healthy or otherwisenon-diseased or damaged tissue, and then the selected substrates aresubsequently counter-screened against proteases expressed in normal,e.g., non-diseased, tissue.

In some embodiments, the antibody or antigen-binding fragment thereof inthe activatable anti-EGFR antibody is derived from the anti-EGFRantibody cetuximab. Cetuximab, also known as Erbitux or C225, is achimeric (mouse/human) monoclonal antibody that specifically binds EGFR.Cetuximab is currently used in the treatment of metastatic colorectalcancer and head and neck cancer.

Preferably, the activatable anti-EGFR antibody includes a heavy chainthat is or is derived from the amino acid sequence of SEQ ID NO: 26, theamino acid sequence of SEQ ID NO: 30, or the amino acid sequence of SEQID NO: 34.

In some embodiments, the sequence of the antibody or antigen-bindingfragment thereof that binds EGFR contains at least one amino acidsubstitution to remove a potential site of glycosylation. For example,the antibody or antigen-binding fragment thereof that binds EGFRcontains an amino acid substitution in the heavy chain and/or the lightchain to remove a potential site of glycosylation. In some embodiments,the antibody or antigen-binding fragment thereof that binds EGFR has aheavy chain in which the asparagine (N) corresponding to the asparagineat position 88 of the heavy chain having amino acid sequence of SEQ IDNO: 30 is replaced with a glutamine (Q) residue to yield a heavy chainhaving the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the antibody or antigen-binding fragment thereofthat binds EGFR in the activatable antibodies can include additionalmodifications, particularly in the Fc region of the antibody orantigen-binding fragment thereof. For example, the Fc region of theantibody or antigen-binding fragment thereof can include one or moreamino acid substitutions to remove a potential site of glycosylation andor to disrupt binding of the Fc region of its receptor. In someembodiments, the antibody or antigen-binding fragment thereof that bindsEGFR has a heavy chain in which the asparagine (N) corresponding to theasparagine at position 299 of the heavy chain having amino acid sequenceof SEQ ID NO: 30 is replaced with an alanine (A) residue. This mutationremoves a glycosylation site in the Fc region and leads to reduced Fcreceptor binding by the Fc. In some embodiments, the antibody orantigen-binding fragment thereof that binds EGFR has a heavy chain inwhich the asparagine (N) corresponding to the asparagine at position 88of the heavy chain having amino acid sequence of SEQ ID NO: 30 isreplaced with a glutamine (Q) residue and in which the asparagine (N)corresponding to the asparagine at position 299 of the heavy chainhaving amino acid sequence of SEQ ID NO: 30 is replaced with an alanine(A) residue to yield a heavy chain having the amino acid sequence of SEQID NO: 34.

In some embodiments, the activatable anti-EGFR antibody includes asignal peptide. The signal peptide can be linked to the activatableanti-EGFR antibody by a spacer. In some embodiments, the spacer isconjugated to the activatable antibody in the absence of a signalpeptide. In some embodiments, the spacer is joined directly to the MM ofthe activatable antibody. In some embodiments, the spacer has amino acidsequence QGQSGQ (SEQ ID NO: 38). In some embodiments, an activatableantibody comprises a spacer of sequence QGQSGQ (SEQ ID NO: 38) joineddirectly to a MM sequence CISPRGCPDGPYVMY (SEQ ID NO: 14) in thestructural arrangement from N-terminus to C-terminus of spacer-MM-CM-AB.In some embodiments, an activatable antibody includes a spacer joineddirectly to a MM sequence and includes the amino acid sequenceQGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 59) in the structural arrangement fromN-terminus to C-terminus of spacer-MM-CM-AB.

The activatable anti-EGFR antibodies provided herein include a maskingmoiety. In some embodiments, the masking moiety is an amino acidsequence that is coupled, or otherwise attached, to the anti-EGFRantibody and is positioned within the activatable anti-EGFR antibodyconstruct such that the masking moiety reduces the ability of theanti-EGFR antibody to specifically bind EGFR. Suitable masking moietiesare identified using any of a variety of known techniques. For example,peptide masking moieties are identified using the methods described inU.S. Patent Application Publication No. 2009/0062142 by Daugherty etal., the contents of which are hereby incorporated by reference in theirentirety.

The activatable anti-EGFR antibodies provided herein include a cleavablemoiety. In some embodiments, the cleavable moiety includes an amino acidsequence that is a substrate for a protease, usually an extracellularprotease. Suitable substrates are identified using any of a variety ofknown techniques. For example, peptide substrates are identified usingthe methods described in U.S. Pat. No. 7,666,817 by Daugherty et al.,the contents of which are hereby incorporated by reference in theirentirety. (See also Boulware et al. “Evolutionary optimization ofpeptide substrates for proteases that exhibit rapid hydrolysiskinetics.” Biotechnol Bioeng. 106.3 (2010): 339-46).

The activatable anti-EGFR antibodies described herein overcome alimitation of antibody therapeutics, particularly antibody therapeuticsthat are known to be toxic to at least some degree in vivo.Target-mediated toxicity constitutes a major limitation for thedevelopment of therapeutic antibodies. This is best exemplified by theskin rash that afflicts 88% of patients treated with cetuximab, anantibody that specifically binds epidermal growth factor receptor (EGFR)and has been approved for the treatment of colorectal and head and neckcancer. The activatable anti-EGFR antibodies provided herein aredesigned to address the toxicity associated with the inhibition of thetarget in normal tissues by traditional therapeutic antibodies. Theseactivatable anti-EGFR antibodies remain masked until proteolyticallyactivated at the site of disease. Starting with cetuximab as a parentaltherapeutic antibody, the activatable anti-EGFR antibodies of theinvention were engineered by coupling the antibody to an inhibitory maskthrough a linker that incorporates a protease substrate. In studiesperformed in vitro studies, the binding to EGFR and the cell-basedactivity of the activatable anti-EGFR antibody was diminished comparedto cetuximab. In studies performed in vivo studies, the activatableanti-EGFR antibody remained masked in normal tissues, but was activatedand accumulated in the tumor environment. The tumor activation of theactivatable anti-EGFR antibody translated into an in vivo efficacy thatwas equivalent to the efficacy of cetuximab.

The activatable anti-EGFR antibodies provided herein satisfy asignificant clinical need. EGFR is a clinically validated target thathas been shown to promote proliferation, angiogenesis andinvasion/metastasis, as well as to inhibit apoptosis of tumor cells.Antibodies and small molecule tyrosine kinase inhibitors targeting EGFRhave been approved for cancer treatment (Nature Rev Cancer 5 (2005) 341;Curr Opin Cell Biol 21, (2009) 177). However, current anti-EGFRtherapies, also referred to as EGFR inhibitors (EGFRi), have been shownto exhibit a number of adverse events post-treatment, including forexample, papulopustular rash, particularly in the face and upper trunkof human subjects; dry and itchy skin; inflammation around the nails,loss of hair on the scalp; and increased growth of eyelashes and facialhair. Cutaneous toxicities that results from treatment with EGFRi havebeen shown to affect 45-100% of patients. (See e.g., Segaert and VanCutsem. Ann Oncol. 16(9) (2005):1425-33).

Exemplary activatable anti-EGFR antibodies of the invention include, forexample, the activatable antibody referred to herein as the3954-1204-C225v5 activatable antibody, which binds epidermal growthfactor receptor (EGFR). Two sequences of the 3954-1204-C225v5activatable anti-EGFR antibody are shown below, Sequence 1 is thesequence of a version of the 3954-1204-C225v5 activatable anti-EGFRantibody that includes a signal peptide, and Sequence 2 is the sequenceof the 3954-1204-C225v5 activatable anti-EGFR antibody without thesignal peptide:

3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 60)][C225v5 (SEQ ID NO: 25)] (SEQ ID NO: 1)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 61)][C225v5 (SEQ ID NO: 26)] (SEQ ID NO: 2)[MYRMQLLSCI ALSLALVTNS] [QVQLKQSGPG LVQPSQSLSI TCTVSGFSLTNYGVHWVRQS PGKGLEWLGV IWSGGNTDYN TPFTSRLSIN KDNSKSQVFFKMNSLQSQDT AIYYCARALT YYDYEFAYWG QGTLVTVSAA STKGPSVFPLAPSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK*] Italics: Signal peptideNormal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 60)][Spacer (SEQ ID NO: 62)][Mask(SEQ ID NO: 63)][Linker 1 (SEQ ID NO: 64)][1204 Substrate (SEQID NO: 65)][Linker 2 (SEQ ID NO: 66)][C225 (SEQ ID NO: 67)](SEQ ID NO: 3)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac][ggctcgagcggtggcagcggtggctctggtggatccggt][ctgagcggccgttccgata atcat][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 61)][Spacer (SEQ ID NO: 38)][Mask(SEQ ID NO: 14)][Linker 1 (SEQ ID NO: 23)][1204 Substrate (SEQID NO: 13)][Linker 2 (SEQ ID NO: 24)][C225 (SEQ ID NO: 68)](SEQ ID NO: 4) [MYRMQLLSCI ALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGS GGSG ][ LSGRSDNH ][

][QIL LTQSPVILSV SPGERVSFSC RASQSIGTNIHWYQQRTNGS PRLLIKYASE SISGIPSRFS GSGSGTDFTL SINSVESEDIADYYCQQNNN WPTTFGAGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVVCLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSKADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*] Italics: Signal peptideBold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v5 (SEQ ID NO: 25)] (SEQ ID NO: 25)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga]3954-1204-C225v5 Activatable Antibody Heavy Chain Amino Acid Sequence 2:[C225v5 (SEQ ID NO: 26)] (SEQ ID NO: 26)[QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT NYGVHWVRQS PGKGLEWLGVIWSGGNTDYN TPFTSRLSIN KDNSKSQVFF KMNSLQSQDT AIYYCARALTYYDYEFAYWG QGTLVTVSAA STKGPSVFPL APSSKSTSGG TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTYICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQVYTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK*]3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 62)][Mask (SEQ ID NO: 63)][Linker 1 (SEQ IDNO: 64)][1204 Substrate (SEQ ID NO: 65)][Linker 2 (SEQ ID NO:66)][C225 (SEQ ID NO: 67)] (SEQ ID NO: 27)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtacggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v5 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 38)][Mask (SEQ ID NO: 14)][Linker 1 (SEQ IDNO: 23)][1204 Substrate (SEQ ID NO: 13)][Linker 2 (SEQ ID NO:24)][C225 (SEQ ID NO: 68)] (SEQ ID NO: 28) [QGQSGQ][CISP RGCPDGPYVMY][GSSGGSGGSGGSG ][ LSGRSDNH ][

][QIL LTQSPVILSV SPGERVSFSC RASQSIGTNI HWYQQRTNGS PRLLIKYASESISGIPSRFS GSGSGTDFTL SINSVESEDI ADYYCQQNNN WPTTFGAGTKLELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNALQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*]Bold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Another exemplary activatable anti-EGFR antibody of the invention is theactivatable antibody referred to herein as the 3954-1204-C225v4activatable antibody, which binds epidermal growth factor receptor(EGFR). Two sequences of the 3954-1204-C225v4 activatable anti-EGFRantibody are shown below, Sequence 1 is the sequence of a version of the3954-1204-C225v4 activatable anti-EGFR antibody that includes a signalpeptide, and Sequence 2 is the sequence of the 3954-1204-C225v4activatable anti-EGFR antibody without the signal peptide:

3954-1204-C225v4 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal Peptide (SEQ ID NO: 60)][C225v4 (SEQ ID NO: 29)] (SEQ ID NO: 5)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal Peptide (SEQ ID NO: 61)][C225v4 (SEQ ID NO: 30)] (SEQ ID NO: 6)[MYRMQLLSCI ALSLALVTNS][QVQLKQSGPG LVQPSQSLSI TCTVSGFSLTNYGVHWVRQS PGKGLEWLGV IWSGGNTDYN TPFTSRLSIN KDNSKSQVFFKMNSLQSNDT AIYYCARALT YYDYEFAYWG QGTLVTVSAA STKGPSVFPLAPSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK*] Italics: Signal peptideNormal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 60)][Spacer (SEQ ID NO: 62)][Mask(SEQ ID NO: 63)][Linker 1 (SEQ ID NO: 64)][1204 Substrate (SEQID NO: 65)][Linker 2 (SEQ ID NO: 66)][C225 (SEQ ID NO: 67)](SEQ ID NO: 3)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 61)][Spacer (SEQ ID NO: 38)][Mask(SEQ ID NO: 14)][Linker 1 (SEQ ID NO: 23)][1204 Substrate (SEQID NO: 13)][Linker 2 (SEQ ID NO: 24)][C225 (SEQ ID NO: 68)](SEQ ID NO: 4) [MYRMQLLSCI ALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGS GGSG ][ LSGRSD   NH ][

][QIL LTQSPVILSVSPGERVSFSC RASQSIGTNIHWYQQRTNGS PRLLIKYASE SISGIPSRFS GSGSGTDFTL SINSVESEDIADYYCQQNNN WPTTFGAGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVVCLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSKADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*] Italics: Signal peptideBold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v4 (SEQ ID NO: 29)] (SEQ ID NO: 29)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga]3954-1204-C225v4 Activatable Antibody Heavy Chain Amino Acid Sequence 2:[C225v4 (SEQ ID NO: 30)] (SEQ ID NO: 30)[QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT NYGVHWVRQS PGKGLEWLGVIWSGGNTDYN TPFTSRLSIN KDNSKSQVFF KMNSLQSNDT AIYYCARALTYYDYEFAYWG QGTLVTVSAA STKGPSVFPL APSSKSTSGG TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTYICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQVYTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK*]3954-1204-C225v4 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 62)][Mask (SEQ ID NO: 63)][Linker 1 (SEQ IDNO: 64)][1204 Substrate (SEQ ID NO: 65)][Linker 2 (SEQ ID NO:66)][C225 (SEQ ID NO: 67)] (SEQ ID NO: 27)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v4 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 38)][Mask (SEQ ID NO: 14)][Linker 1 (SEQ IDNO: 23)][1204 Substrate (SEQ ID NO: 13)][Linker 2 (SEQ ID NO:24)][C225 (SEQ ID NO: 68)] (SEQ ID NO: 28) [QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGSGGSG ][ LSGRSDNH ][

][QIL LTQSPVILSV SPGERVSFSC RASQSIGTNI HWYQQRTNGS PRLLIKYASESISGIPSRFS GSGSGTDFTL SINSVESEDI ADYYCQQNNN WPTTFGAGTKLELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNALQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*]Bold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

Another exemplary activatable anti-EGFR antibody of the invention is theactivatable antibody referred to herein as the 3954-1204-C225v6activatable antibody, which binds epidermal growth factor receptor(EGFR). Two sequences of the 3954-1204-C225v6 activatable anti-EGFRantibody are shown below, Sequence 1 is the sequence of a version of the3954-1204-C225v6 activatable anti-EGFR antibody that includes a signalpeptide, and Sequence 2 is the sequence of the 3954-1204-C225v6activatable anti-EGFR antibody without the signal peptide:

3954-1204-C225v6 Activatable Antibody Heavy Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 60)][C225v6 (SEQ ID NO: 33)] (SEQ ID NO: 9)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggta aatga]Italics: Signal peptide Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Heavy Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 61)][C225v6 (SEQ ID NO: 34)] (SEQ ID NO: 10)[MYRMQLLSCI ALSLALVTNS][QVQLKQSGPG LVQPSQSLSI TCTVSGFSLTNYGVHWVRQS PGKGLEWLGV IWSGGNTDYN TPFTSRLSIN KDNSKSQVFFKMNSLQSQDT AIYYCARALT YYDYEFAYWG QGTLVTVSAA STKGPSVFPLAPSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYAS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK*] Italics: Signal peptideNormal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Nucleotide Sequence 1:[Signal peptide (SEQ ID NO: 60)][Spacer (SEQ ID NO: 62)][Mask(SEQ ID NO: 63)][Linker 1 (SEQ ID NO: 64)][1204 Substrate (SEQID NO: 65)][Linker 2 (SEQ ID NO: 66)][C225 (SEQ ID NO: 67)](SEQ ID NO: 3)[atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg][ caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgtac] [ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgataat cat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Italics: Signal peptide Bold: SpacerUnderline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Amino Acid Sequence 1:[Signal peptide (SEQ ID NO: 61)][Spacer (SEQ ID NO: 38)][Mask(SEQ ID NO: 14)][Linker 1 (SEQ ID NO: 23)][1204 Substrate (SEQID NO: 13)][Linker 2 (SEQ ID NO: 24)][C225 (SEQ ID NO: 68)](SEQ ID NO: 4) [MYRMQLLSCI ALSLALVTNS][QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGS GGSG ][ LSGRSDNH ][

][QILLTQSPVILSV SPGERVSFSC RASQSIGTNIHWYQQRTNGS PRLLIKYASE SISGIPSRFS GSGSGTDFTL SINSVESEDIADYYCQQNNN WPTTFGAGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVVCLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSKADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*] Italics: Signal peptideBold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Heavy Chain Nucleotide Sequence 2:[C225v6 (SEQ ID NO: 33)] (SEQ ID NO: 33)[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga]3954-1204-C225v6 Activatable Antibody Heavy Chain Amino Acid Sequence 2:[C225v6 (SEQ ID NO: 34)] (SEQ ID NO: 34)[QVQLKQSGPG LVQPSQSLSI TCTVSGFSLT NYGVHWVRQS PGKGLEWLGVIWSGGNTDYN TPFTSRLSIN KDNSKSQVFF KMNSLQSQDT AIYYCARALTYYDYEFAYWG QGTLVTVSAA STKGPSVFPL APSSKSTSGG TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTYICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYASTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQVYTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK*]3954-1204-C225v6 Activatable Antibody Light Chain Nucleotide Sequence 2:[Spacer (SEQ ID NO: 62)][Mask (SEQ ID NO: 63)][Linker 1 (SEQ IDNO: 64)][1204 Substrate (SEQ ID NO: 65)][Linker 2 (SEQ IDNO: 66)][C225 (SEQ ID NO: 67)] (SEQ ID NO: 27)[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacggcccatacgtcatgt ac][ggctcgagcggtggcagcggtggctctggtggatccggt ][ ctgagcggccgttccgata atcat ][

][cagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: MaskItalics and Underline: Linker 1 Bold and Underline: 1204 SubstrateBold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence3954-1204-C225v6 Activatable Antibody Light Chain Amino Acid Sequence 2:[Spacer (SEQ ID NO: 38)][Mask (SEQ ID NO: 14)][Linker 1 (SEQ IDNO: 23)][1204 Substrate (SEQ ID NO: 13)][Linker 2 (SEQ IDNO: 24)][C225 (SEQ ID NO: 68)] (SEQ ID NO: 28)[QGQSGQ][CISPRGCPDGPYVMY][ GSSGGSGGSGGSG ][ LSGRSDNH ][

][QIL LTQSPVILSV SPGERVSFSC RASQSIGTNI HWYQQRTNGS PRLLIKYASESISGIPSRFS GSGSGTDFTL SINSVESEDI ADYYCQQNNN WPTTFGAGTKLELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNALQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C*]Bold: Spacer Underline: Mask Italics and Underline: Linker 1Bold and Underline: 1204 Substrate Bold, Italics and Underline: Linker 2Normal text: anti-EGFR antibody derived sequence

When an activatable anti-EGFR antibody of the invention is cleaved by aprotease, i.e., when the activatable anti-EGFR is in an active orcleaved state, the activated anti-EGFR antibody will retain only aportion of the amino acid sequence of the activatable antibody in aninactive or uncleaved state. The sequence of the activatable anti-EGFRin an active or cleaved state will vary depending on which proteasecleaves the substrate (CM), as different proteases can have differentrecognition sites.

For example, the exemplary anti-EGFR antibodies of the invention, the3954-1204-C225v5 activatable antibody, the 3954-1204-C225v4 activatableantibody, and/or the 3954-1204-C225v6 activatable antibody, each havethe same light chain amino acid (referred to herein as Light Chain AminoAcid Sequence 2). When exposed to and activated by either urokinaseplasminogen activator (uPA) or MT-SP1, the activated form of theanti-EGFR activatable antibody would have the following amino acidsequence:

Activated Form of 3954-1204-C225v5, 3954-1204-C225v4 and/or 3954-1204-C225v6Activatable Antibody Light Chain Amino AcidSequence 2 When Activated by uPA or MT-SP1: (SEQ ID NO: 69)SDNHGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* 

When exposed to and activated legumain, the activated form of theanti-EGFR activatable antibody would have the following amino acidsequence:

Activated Form of 3954-1204-C225v5, 3954-1204-C225v4 and/or 3954-1204-C225v6Activatable Antibody Light Chain Amino AcidSequence 2 When Activated by Legumain: (SEQ ID NO: 70)HGSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* 

In some embodiments, the activatable anti-EGFR antibodies describedherein also include an agent conjugated to the activatable antibody. Insome embodiments, the conjugated agent is a therapeutic agent, such asan antineoplastic agent. In such embodiments, the agent is conjugated toa carbohydrate moiety of the activatable antibody, preferably where thecarbohydrate moiety is located outside the antigen-binding region of theantibody or antigen-binding fragment in the activatable antibody. Insome embodiments, the agent is conjugated to a sulfhydryl group of theantibody or antigen-binding fragment in the activatable antibody. Insome embodiments, the agent is conjugated to an amino group of theantibody or antigen-binding fragment of the activatable antibody. Insome embodiments the agent is conjugated to a carboxylic acid group ofthe antibody or antigen-binding fragment of the activatable antibody.

In some embodiments, the agent is a cytotoxic agent such as a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Suitable cytotoxic agents include, for example, any ofthe cytotoxic agents listed in Table 1. In some embodiments, thecytotoxic agent is a dolastatin or a derivative thereof (e.g. auristatinE, AFP, MMAF, MMAE, DMAF, DMAE). For example, the cytotoxic agent ismonomethyl auristatin E (MMAE).

In some embodiments, the conjugated activatable antibody can be modifiedfor site-specific conjugation through modified amino acid sequencesinserted or otherwise included in the activatable antibody sequence.These modified amino acid sequences are designed to allow for controlledplacement and/or dosage of the conjugated agent within a conjugatedactivatable anti-EGFR antibody. For example, the activatable antibodycan be engineered to include cysteine substitutions at positions onlight and heavy chains that provide reactive thiol groups and do notnegatively impact protein folding and assembly, nor alter antigenbinding. In some embodiments, the activatable antibody can be engineeredto include or otherwise introduce one or more non-natural amino acidresidues within the activatable antibody to provide suitable sites forconjugation. In some embodiments, the activatable antibody can beengineered to include or otherwise introduce enzymatically activatablepeptide sequences within the activatable antibody sequence.

In some embodiments, the agent is a detectable moiety such as, forexample, a label or other marker. For example, the agent is or includesa radiolabeled amino acid, one or more biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods), one or more radioisotopes or radionuclides, oneor more fluorescent labels, one or more enzymatic labels, and/or one ormore chemiluminescent agents. In some embodiments, detectable moietiesare attached by spacer molecules.

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Table 1 lists some of the exemplary pharmaceutical agents that may beemployed in the herein described invention but in no way is meant to bean exhaustive list.

TABLE 1 Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTSAuristatins Turbostatin Auristatin E Phenstatins Monomethyl auristatin E(MMAE) Hydroxyphenstatin Desmethyl auristatin E (DMAE) Spongistatin 5Auristatin F Spongistatin 7 Monomethyl auristatin F (MMAF) Halistatin 1Desmethyl auristatin F (DMAF) Halistatin 2 Auristatin derivatives, e.g.,Halistatin 3 amides thereof Auristatin tyramine Modified BryostatinsAuristatin quinoline Halocomstatins Dolastatins Pyrrolobenzimidazoles(PBI) Dolastatin derivatives Cibrostatin6 Dolastatin 16 DmJ DoxaliformDolastatin 16 Dpv Anthracyclins analogues Maytansinoids, e.g. DM-1; DM-4Anthracyclins analogues Maytansinoid derivatives Duocarmycin Cemadotinanalogue (CemCH2-SH) Duocarmycin derivatives Pseudomonas toxin A (PE38)variant Alpha-amanitin Pseudomonas toxin A (ZZ-PE38) variantAnthracyclines ZJ-101 Doxorubicin OSW-1 Daunorubicin4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine BryostatinsTopoisomerase inhibitors Camptothecin Hemiasterlin Camptothecinderivatives Cephalotaxine 7-substituted Camptothecin Homoharringtonine10, 11- Pyrrolobenzodiazepine dimers Difluoromethylenedioxy- (PBDs)camptothecin Combretastatins Functionalized pyrrolobenzodiazepenesDebromoaplysiatoxin Calicheamicins Kahalalide-F PodophyllotoxinsDiscodermolide Taxanes Ecteinascidins Vinca alkaloids CONJUGATABLEDETECTION ANTIVIRALS REAGENTS Acyclovir Fluorescein and derivativesthereof Vira A Fluorescein isothiocyanate (FITC) Symmetrel ANTIFUNGALSNystatin ADDITIONAL ANTI- RADIO- NEOPLASTICS PHARMACEUTICALS Adriamycin¹²⁵I Cerubidine ¹³¹I Bleomycin ⁸⁹Zr Alkeran ¹¹¹In Velban ¹²³I Oncovin¹³¹I Fluorouracil ⁹⁹mTc Methotrexate ²⁰¹Tl Thiotepa ¹³³xe Bisantrene ¹¹CNovantrone ⁶²Cu Thioguanine ¹⁸F Procarabizine ⁶⁸Ga Cytarabine ¹³NANTI-BACTERIALS ¹⁵O Aminoglycosides ³⁸K Streptomycin ⁸²Rb Neomycin ⁹⁹mTc(Technetium) Kanamycin HEAVY METALS Amikacin Barium Gentamicin GoldTobramycin Platinum Streptomycin B ANTI-MYCOPLASMALS SpectinomycinTylosine Ampicillin Spectinomycin Sulfanilamide PolymyxinChloramphenicol

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, the linker SMPT contains a sterically hindereddisulfide bond, and can form conjugates with increased stability.Disulfide linkages, are in general, less stable than other linkagesbecause the disulfide linkage is cleaved in vitro, resulting in lessconjugate available.

The reagent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride is useful to create a carboxamide starting with acarboxylic acid and a primary or secondary amine. Thus, EDC may be usedto link lysine residues in an antibody with a carboxylic acid in alinker or toxin, or to link aspartate or glutamate residues in anantibody with an amine in a linker or toxin. Such conjugation reactionsutilizing EDC may be enhanced by addition of NHS (N-hydroxysuccinimide)or sulfo-NHS (N-hydroxy-3-oxysulfonylsuccinimide). Addition of NHS orsulfo-NHS to such conjugation reactions may enhance the rate,completeness, selectivity, and/or reproducibility of the conjugationreactions.

In some embodiments, the linkers are cleavable. In some embodiments, thelinkers are non-cleavable. In some embodiments, two or more linkers arepresent. The two or more linkers are all the same, e.g., cleavable ornon-cleavable, or the two or more linkers are different, e.g., at leastone cleavable and at least one non-cleavable.

The present invention utilizes several methods for attaching agents toABs: (a) attachment to the carbohydrate moieties of the AB, or (b)attachment to sulfhydryl groups of the AB, or (c) attachment to aminogroups of the AB, or (d) attachment to carboxylate groups of the AB.According to the invention, ABs may be covalently attached to an agentthrough an intermediate linker having at least two reactive groups, oneto react with AB and one to react with the agent. The linker, which mayinclude any compatible organic compound, can be chosen such that thereaction with AB (or agent) does not adversely affect AB reactivity andselectivity. Furthermore, the attachment of linker to agent might notdestroy the activity of the agent. Suitable linkers for reaction withoxidized antibodies or oxidized antibody fragments include thosecontaining an amine selected from the group consisting of primary amine,secondary amine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present invention, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the primary amino groups present inunmodified lysine residues in the Ab. Such reactive groups include, butare not limited to, NHS carboxylic or carbonic esters, sulfo-NHScarboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonicesters, pentafluorophenyl carboxylic or carbonic esters, acylimidazoles, isocyanates, and isothiocyanates.

According to the present invention, suitable linkers for attachment toneither oxidized nor reduced Abs include those having certain functionalgroups capable of reaction with the carboxylic acid groups present inaspartate or glutamate residues in the Ab, which have been activatedwith suitable reagents. Suitable activating reagents include EDC, withor without added NHS or sulfo-NHS, and other dehydrating agents utilizedfor carboxamide formation. In these instances, the functional groupspresent in the suitable linkers would include primary and secondaryamines, hydrazines, hydroxylamines, and hydrazides.

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker. In otherembodiments the AB is first attached to the MM, CM and associatedlinkers and then attached to the linker for conjugation purposes.

Branched Linkers: In specific embodiments, branched linkers that havemultiple sites for attachment of agents are utilized. For multiple sitelinkers, a single covalent attachment to an AB would result in anAB-linker intermediate capable of binding an agent at a number of sites.The sites may be aldehyde or sulfhydryl groups or any chemical site towhich agents can be attached.

Alternatively, higher specific activity (or higher ratio of agents toAB) can be achieved by attachment of a single site linker at a pluralityof sites on the AB. This plurality of sites may be introduced into theAB by either of two methods. First, one may generate multiple aldehydegroups and/or sulfhydryl groups in the same AB. Second, one may attachto an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

Cleavable Linkers: Peptide linkers which are susceptible to cleavage byenzymes of the complement system, such as but not limited to urokinase,tissue plasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentinvention. According to one method of the present invention, an agent isattached via a linker susceptible to cleavage by complement. Theantibody is selected from a class that can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present invention, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasminogen activator, plasmin, or trypsin. These cleavablelinkers are useful in conjugated activatable antibodies that include anextracellular toxin, e.g., by way of non-limiting example, any of theextracellular toxins shown in Table 1.

Non-liming examples of cleavable linker sequences are provided in Table2.

TABLE 2 Exemplary Linker Sequences for Conjugation Types of CleavableSequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinasePRFKIIGG (SEQ ID NO: 7) PRFRIIGG (SEQ ID NO: 8) TGFβSSRHRRALD (SEQ ID NO: 11) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 12)Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 31) SSSFDKGKYKRGDDA (SEQ IDNO: 32) Factor Xa cleavable IEGR (SEQ ID NO: 35) sequencesIDGR (SEQ ID NO: 36) GGSIDGR (SEQ ID NO: 39) MMP cleavable sequencesGelatinase A PLGLWA (SEQ ID NO: 40) Collagenase cleavable sequencesCalf skin collagen GPQGIAGQ (SEQ ID NO: 41) (α1(I) chain)Calf skin collagen GPQGLLGA (SEQ ID NO: 42) (α2(I) chain)Bovine cartilage GIAGQ (SEQ ID NO: 43) collagen (α1(II) chain)Human liver collagen GPLGIAGI (SEQ ID NO: 44) (α1(III) chain) Human α₂MGPEGLRVG (SEQ ID NO: 45) Human PZP YGAGLGVV (SEQ ID NO: 46)AGLGVVER (SEQ ID NO: 47) AGLGISST (SEQ ID NO: 48) Rat α₁MEPQALAMS (SEQ ID NO: 49) QALAMSAI (SEQ ID NO: 50) Rat α₂MAAYHLVSQ (SEQ ID NO: 51) MDAFLESS (SEQ ID NO: 52) Rat α₁I₃(2J)ESLPVVAV (SEQ ID NO: 53) Rat α₁I₃(27J) SAPAVESE (SEQ ID NO: 54)Human fibroblast DVAQFVLT (SEQ ID NO: 55) collagenaseVAQFVLTE (SEQ ID NO: 56) (autolytic cleavages) AQFVLTEG (SEQ ID NO: 57)PVQPIGPQ (SEQ ID NO: 58)

In addition, agents may be attached via disulfide bonds (for example,the disulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In certain specific embodiments the reducing agentthat would modify a CM would also modify the linker of the conjugatedactivatable antibody.

Spacers and Cleavable Elements: In still another embodiment, it may benecessary to construct the linker in such a way as to optimize thespacing between the agent and the AB of the activatable antibody. Thismay be accomplished by use of a linker of the general structure:

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In still other embodiments, the linker may comprise a spacer element anda cleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be particular mode of attachment orreaction. Any reaction providing a product of suitable stability andbiological compatibility is acceptable.

Serum Complement and Selection of Linkers: According to one method ofthe present invention, when release of an agent is desired, an AB thatis an antibody of a class which can activate complement is used. Theresulting conjugate retains both the ability to bind antigen andactivate the complement cascade. Thus, according to this embodiment ofthe present invention, an agent is joined to one end of the cleavablelinker or cleavable element and the other end of the linker group isattached to a specific site on the AB. For example, if the agent has anhydroxy group or an amino group, it may be attached to the carboxyterminus of a peptide, amino acid or other suitably chosen linker via anester or amide bond, respectively. For example, such agents may beattached to the linker peptide via a carbodimide reaction. If the agentcontains functional groups that would interfere with attachment to thelinker, these interfering functional groups can be blocked beforeattachment and deblocked once the product conjugate or intermediate ismade. The opposite or amino terminus of the linker is then used eitherdirectly or after further modification for binding to an AB which iscapable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the activatable antibody. The other end of the linker or spacerelement may be attached to an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond which attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 1.

Linkers for Release without Complement Activation: In yet anotherapplication of targeted delivery, release of the agent withoutcomplement activation is desired since activation of the complementcascade will ultimately lyse the target cell. Hence, this approach isuseful when delivery and release of the agent should be accomplishedwithout killing the target cell. Such is the goal when delivery of cellmediators such as hormones, enzymes, corticosteroids, neurotransmitters,genes or enzymes to target cells is desired. These conjugates may beprepared by attaching the agent to an AB that is not capable ofactivating complement via a linker that is mildly susceptible tocleavage by serum proteases. When this conjugate is administered to anindividual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

Biochemical Cross Linkers: In other embodiments, the activatableantibody may be conjugated to one or more therapeutic agents usingcertain biochemical cross-linkers. Cross-linking reagents form molecularbridges that tie together functional groups of two different molecules.To link two different proteins in a step-wise manner,hetero-bifunctional cross-linkers can be used that eliminate unwantedhomopolymer formation.

Peptidyl linkers cleavable by lysosomal proteases are also useful, forexample, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labilelinkers cleavable in the low-pH environment of the lysosome may be used,for example: bis-sialyl ether. Other suitable linkers includecathepsin-labile substrates, particularly those that show optimalfunction at an acidic pH.

Exemplary hetero-bifunctional cross-linkers are referenced in Table 3.

TABLE 3 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length after crosslinking- Reactive (Ang-Linker Toward Advantages and Applications stroms) SMPT Primary aminesGreater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation  6.8Å Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extendedspacer arm 15.6 Å Sulfhydryls Sulfo-LC- Primary amines Extender spacerarm 15.6 Å SPDP Sulfhydryls Water-soluble SMCC Primary amines Stablemaleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugationHapten-carrier protein conjugation Sulfo- Primary amines Stablemaleimide reactive 11.6 Å SMCC group Sulfhydryls Water-solubleEnzyme-antibody conjugation MBS Primary amines Enzyme-antibodyconjugation  9.9 Å Sulfhydryls Hapten-carrier protein conjugation Sulfo-Primary amines Water-soluble  9.9 Å MBS Sulfhydryls SIAB Primary aminesEnzyme-antibody conjugation 10.6 Å Sulfhydryls Sulfo- Primary aminesWater-soluble 10.6 Å SIAB Sulfhydryls SMPB Primary amines Extendedspacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo- Primaryamines Extended spacer arm 14.5 Å SMPB Sulfhydryls Water-soluble EDE/Primary amines Hapten-Carrier conjugation 0 Sulfo- Carboxyl groups NHSABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective

Non-Cleavable Linkers or Direct Attachment: In still other embodimentsof the invention, the conjugate may be designed so that the agent isdelivered to the target but not released. This may be accomplished byattaching an agent to an AB either directly or via a non-cleavablelinker.

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds which may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A-general formula for such an organiclinker could be

W—(CH₂)n-Q

whereinW is either —NH—CH₂— or —CH₂—;Q is an amino acid, peptide; andn is an integer from 0 to 20.

Non-Cleavable Conjugates: Alternatively, a compound may be attached toABs which do not activate complement. When using ABs that are incapableof complement activation, this attachment may be accomplished usinglinkers that are susceptible to cleavage by activated complement orusing linkers that are not susceptible to cleavage by activatedcomplement.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, Fab,and F(ab′)₂ fragments, scFvs, and an Fab expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≤1 μM; preferably ≤100 nM and most preferably ≤10 nM.

As used herein, the terms “specific binding,” “immunological binding,”and “immunological binding properties” refer to the non-covalentinteractions of the type which occur between an immunoglobulin moleculeand an antigen for which the immunoglobulin is specific. The strength,or affinity of immunological binding interactions can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. Immunological bindingproperties of selected polypeptides can be quantified using methods wellknown in the art. One such method entails measuring the rates ofantigen-binding site/antigen complex formation and dissociation, whereinthose rates depend on the concentrations of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the “on rate constant”(K_(on)) and the “off rate constant” (K_(off)) can be determined bycalculation of the concentrations and the actual rates of associationand dissociation. (See Nature 361:186-87 (1993)). The ratio ofK_(off)/K_(on) enables the cancellation of all parameters not related toaffinity, and is equal to the dissociation constant K_(d). (See,generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). Anantibody of the present invention is said to specifically bind to EGFR,when the equilibrium binding constant (K_(d)) is ≤1 μM, preferably ≤100nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM,as measured by assays such as radioligand binding assays or similarassays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein meansnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotide linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion and/or oneor more internal deletion(s), but where the remaining amino acidsequence is identical to the corresponding positions in thenaturally-occurring sequence deduced, for example, from a full lengthcDNA sequence. Fragments typically are at least 5, 6, 8 or 10 aminoacids long, preferably at least 14 amino acids long′ more preferably atleast 20 amino acids long, usually at least 50 amino acids long, andeven more preferably at least 70 amino acids long. The term “analog” asused herein refers to polypeptides which are comprised of a segment ofat least 25 amino acids that has substantial identity to a portion of adeduced amino acid sequence and which has specific binding to EGFR,under suitable binding conditions. Typically, polypeptide analogscomprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. Analogstypically are at least 20 amino acids long, preferably at least 50 aminoacids long or longer, and can often be as long as a full-lengthnaturally-occurring polypeptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibodies

Activatable antibodies of the invention specifically bind humanepidermal growth factor receptor (EGFR). Also included in the inventionare activatable antibodies that bind to the same epitope as theactivatable anti-EGFR antibodies described herein.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., a murine monoclonal or humanized antibody) has the samespecificity as a monoclonal antibody used in the methods describedherein by ascertaining whether the former prevents the latter frombinding to EGFR. If the monoclonal antibody being tested competes withthe monoclonal antibody of the invention, as shown by a decrease inbinding by the monoclonal antibody of the invention, then the twomonoclonal antibodies bind to the same, or a closely related, epitope.An alternative method for determining whether a monoclonal antibody hasthe specificity of a monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with EGFR and thenadd the monoclonal antibody being tested to determine if the monoclonalantibody being tested is inhibited in its ability to bind EGFR. If themonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the invention.

Use of Activatable Anti-EGFR Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include an activatableanti-EGFR antibody, are used to prevent, treat or otherwise ameliorate adisease or disorder associated with aberrant EGFR expression and/oractivity. For example, therapeutic formulations of the invention, whichinclude an activatable anti-EGFR antibody, are used to treat orotherwise ameliorate a cancer or other neoplastic condition.

Increased proteolysis is known to be a hallmark of cancer. (See e.g.,Affara N I, et al. “Delineating protease functions during cancerdevelopment.” Methods Mol Biol. 539 (2009): 1-32). Progression, invasionand metastasis of tumors result from several interdependent processes inwhich proteases are implicated. This process is shown generally in FIGS.8A and 8B.

Efficaciousness of prevention, amelioration or treatment is determinedin association with any known method for diagnosing or treating thedisease or disorder associated with aberrant EGFR expression and/oractivity. Prolonging the survival of a subject or otherwise delaying theprogression of the disease or disorder associated with aberrant EGFRexpression and/or activity in a subject indicates that the activatableantibody confers a clinical benefit.

Activatable anti-EGFR antibodies can be administered in the form ofpharmaceutical compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) MackPub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,Possibilities, Limitations, And Trends, Harwood Academic Publishers,Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Where activatable antibody fragments are used, the smallest fragmentthat specifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

In some embodiments, the activatable antibody contains a detectablelabel. An intact antibody, or a fragment thereof (e.g., Fab, scFv, orF(ab)₂) is used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, immunochemical staining, and immunofluorescence.In vitro techniques for detection of an analyte genomic DNA includeSouthern hybridizations. Procedures for conducting immunoassays aredescribed, for example in “ELISA: Theory and Practice: Methods inMolecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa,N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, AcademicPress, Inc., San Diego, Calif., 1996; and “Practice and Theory of EnzymeImmunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985.Furthermore, in vivo techniques for detection of an analyte proteininclude introducing into a subject a labeled anti-analyte proteinantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

Diagnostic and Prophylactic Formulations

The anti-EGFR antibodies and/or activatable anti-EGFR antibodies of theinvention are used in diagnostic and prophylactic formulations. In oneembodiment, an anti-EGFR antibody and/or activatable anti-EGFR antibodyis administered to patients that are at risk of developing one or moreof the aforementioned cancer or other disorders. A patient's or organ'spredisposition to one or more of the aforementioned disorders can bedetermined using genotypic, serological or biochemical markers.

In another embodiment of the invention, an anti-EGFR antibody and/oractivatable anti-EGFR antibody is administered to human individualsdiagnosed with a clinical indication associated with one or more of theaforementioned disorders. Upon diagnosis, an anti-EGFR antibody and/oractivatable anti-EGFR antibody is administered to mitigate or reversethe effects of the clinical indication.

Antibodies and/or activatable antibodies of the invention are alsouseful in the detection of EGFR in patient samples and accordingly areuseful as diagnostics. For example, the anti-EGFR antibodies and/oractivatable anti-EGFR antibodies of the invention are used in in vitroassays, e.g., ELISA, to detect EGFR levels in a patient sample.

In one embodiment, an anti-EGFR antibody and/or activatable anti-EGFRantibody of the invention is immobilized on a solid support (e.g., thewell(s) of a microtiter plate). The immobilized antibody and/oractivatable antibody serves as a capture antibody for any EGFR that maybe present in a test sample. Prior to contacting the immobilizedantibody with a patient sample, the solid support is rinsed and treatedwith a blocking agent such as milk protein or albumin to preventnonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of EGFR antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using theanti-EGFR antibodies of the invention in an in vitro diagnostic assay,it is possible to stage a disease in a subject based on expressionlevels of the EGFR antigen. For a given disease, samples of blood aretaken from subjects diagnosed as being at various stages in theprogression of the disease, and/or at various points in the therapeutictreatment of the disease. Using a population of samples that providesstatistically significant results for each stage of progression ortherapy, a range of concentrations of the antigen that may be consideredcharacteristic of each stage is designated.

Anti-EGFR antibodies and/or activatable anti-EGFR antibodies can also beused in diagnostic and/or imaging methods. In some embodiments, suchmethods are in vitro methods. In some embodiments, such methods are invivo methods. In some embodiments, such methods are in situ methods. Insome embodiments, such methods are ex vivo methods. For example,activatable anti-EGFR antibodies having an enzymatically cleavable CMcan be used to detect the presence or absence of an enzyme that iscapable of cleaving the CM. Such activatable anti-EGFR antibodies can beused in diagnostics, which can include in vivo detection (e.g.,qualitative or quantitative) of enzyme activity (or, in someembodiments, an environment of increased reduction potential such asthat which can provide for reduction of a disulfide bond) throughmeasured accumulation of activated anti-EGFR antibodies (i.e.,antibodies resulting from cleavage of an activatable anti-EGFR antibody)in a given cell or tissue of a given host organism. Such accumulation ofactivated anti-EGFR antibodies indicates not only that the tissueexpresses enzymatic activity (or an increased reduction potentialdepending on the nature of the CM) but also that the tissue expressestarget to which the activated antibody binds.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label or radioactivelabel or radiotracer) can be conjugated to an AB or other region of ananti-EGFR antibody and/or activatable anti-EGFR antibody. Suitabledetectable labels are discussed in the context of the above screeningmethods and additional specific examples are provided below. Using an ABspecific to a protein or peptide of the disease state, along with aprotease whose activity is elevated in the disease tissue of interest,activatable anti-EGFR antibodies will exhibit an increased rate ofbinding to disease tissue relative to tissues where the CM specificenzyme is not present at a detectable level or is present at a lowerlevel than in disease tissue or is inactive (e.g., in zymogen form or incomplex with an inhibitor). Since small proteins and peptides arerapidly cleared from the blood by the renal filtration system, andbecause the enzyme specific for the CM is not present at a detectablelevel (or is present at lower levels in non-disease tissues or ispresent in inactive conformation), accumulation of activated anti-EGFRantibodies in the disease tissue is enhanced relative to non-diseasetissues.

In another example, activatable anti-EGFR antibodies can be used todetect the presence or absence of a cleaving agent in a sample. Forexample, where the activatable anti-EGFR antibodies contain a CMsusceptible to cleavage by an enzyme, the activatable anti-EGFRantibodies can be used to detect (either qualitatively orquantitatively) the presence of an enzyme in the sample. In anotherexample, where the activatable anti-EGFR antibodies contain a CMsusceptible to cleavage by reducing agent, the activatable anti-EGFRantibodies can be used to detect (either qualitatively orquantitatively) the presence of reducing conditions in a sample. Tofacilitate analysis in these methods, the activatable antibodies can bedetectably labeled, and can be bound to a support (e.g., a solidsupport, such as a slide or bead). The detectable label can bepositioned on a portion of the activatable anti-EGFR antibody that isnot released following cleavage, for example, the detectable label canbe a quenched fluorescent label or other label that is not detectableuntil cleavage has occurred. The assay can be conducted by, for example,contacting the immobilized, detectably labeled activatable anti-EGFRantibodies with a sample suspected of containing an enzyme and/orreducing agent for a time sufficient for cleavage to occur, then washingto remove excess sample and contaminants. The presence or absence of thecleaving agent (e.g., enzyme or reducing agent) in the sample is thenassessed by a change in detectable signal of the activatable anti-EGFRantibodies prior to contacting with the sample e.g., the presence ofand/or an increase in detectable signal due to cleavage of theactivatable antibody by the cleaving agent in the sample.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe activatable anti-EGFR antibodies when cleaved. Thus, the assays canbe adapted to assess the presence or absence of a cleaving agent and thepresence or absence of a target of interest (EGFR). The presence orabsence of the cleaving agent can be detected by the presence of and/oran increase in detectable label of the activatable anti-EGFR antibodiesas described above, and the presence or absence of the target can bedetected by detection of a target-AB complex e.g., by use of adetectably labeled anti-target antibody.

Activatable anti-EGFR antibodies are also useful in in situ imaging forthe validation of activatable antibody activation, e.g., by proteasecleavage, and binding to a particular target. In situ imaging is atechnique that enables localization of proteolytic activity and targetin biological samples such as cell cultures or tissue sections. Usingthis technique, it is possible to confirm both binding to a given targetand proteolytic activity based on the presence of a detectable label(e.g., a fluorescent label).

These techniques are useful with any frozen cells or tissue derived froma disease site (e.g. tumor tissue) or healthy tissues. These techniquesare also useful with fresh cell or tissue samples.

In these techniques, an activatable anti-EGFR antibody is labeled with adetectable label. The detectable label may be a fluorescent dye, (e.g.Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), anear infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, ahapten, a radioactive marker, biotin and an amplification reagent suchas streptavidin, or an enzyme (e.g., horseradish peroxidase or alkalinephosphatase).

Detection of the label in a sample that has been incubated with thelabeled, activatable anti-EGFR antibody indicates that the samplecontains the target, i.e., EGFR, and contains a protease that isspecific for the CM of the activatable anti-EGFR antibody. In someembodiments, the presence of the protease can be confirmed using broadspectrum protease inhibitors such as those described herein, and/or byusing an agent that is specific for the protease, for example, anantibody such as A11, which is specific for the protease matriptase(MT-SP1) and inhibits the proteolytic activity of MT-SP1; see e.g.,International Publication Number WO 2010/129609, published 11 Nov. 2010.The same approach of using broad spectrum protease inhibitors such asthose described herein, and/or by using a more selective inhibitoryagent can be used to identify a protease or class of proteases specificfor the CM of the activatable anti-EGFR antibody. In some embodiments,the presence of the target can be confirmed using an agent that isspecific for the target, e.g., another anti-EGFR antibody, or thedetectable label can be competed with unlabeled EGFR. In someembodiments, unlabeled activatable anti-EGFR antibody could be used,with detection by a labeled secondary antibody or more complex detectionsystem.

Similar techniques are also useful for in vivo imaging where detectionof the fluorescent signal in a subject, e.g., a mammal, including ahuman, indicates that the disease site contains the target, i.e. EGFR,and contains a protease that is specific for the CM of the activatableanti-EGFR antibody.

These techniques are also useful in kits and/or as reagents for thedetection, identification or characterization of protease activity in avariety of cells, tissues, and organisms based on the protease-specificCM in the activatable anti-EGFR antibody.

In some embodiments, in situ imaging and/or in vivo imaging are usefulin methods to identify which patients to treat. For example, in in situimaging, the activatable anti-EGFR antibodies are used to screen patientsamples to identify those patients having the appropriate protease(s)and target(s) at the appropriate location, e.g., at a tumor site.

In some embodiments in situ imaging is used to identify or otherwiserefine a patient population suitable for treatment with an anti-EGFRactivatable antibody of the disclosure. For example, patients that testpositive for both the target (e.g., EGFR) and a protease that cleavesthe substrate in the cleavable moiety (CM) of the anti-EGFR activatableantibody being tested (e.g., accumulate activated antibodies at thedisease site) are identified as suitable candidates for treatment withsuch an anti-EGFR activatable antibody comprising such a CM. Likewise,patients that test negative for either or both of the target (e.g.,EGFR) and the protease that cleaves the substrate in the CM in theactivatable antibody being tested using these methods are identified assuitable candidates for another form of therapy (i.e., not suitable fortreatment with the anti-EGFR activatable antibody being tested). In someembodiments, such patients that test negative with respect to a firstanti-EGFR activatable antibody can be tested with other anti-EGFRactivatable antibodies comprising different CMs until a suitableanti-EGFR activatable antibody for treatment is identified (e.g., ananti-EGFR activatable antibody comprising a CM that is cleaved by thepatient at the site of disease).

In some embodiments in vivo imaging is used to identify or otherwiserefine a patient population suitable for treatment with an anti-EGFRactivatable antibody of the disclosure. For example, patients that testpositive for both the target (e.g., EGFR) and a protease that cleavesthe substrate in the cleavable moiety (CM) of the anti-EGFR activatableantibody being tested (e.g., accumulate activated antibodies at thedisease site) are identified as suitable candidates for treatment withsuch an anti-EGFR activatable antibody comprising such a CM. Likewise,patients that test negative are identified as suitable candidates foranother form of therapy (i.e., not suitable for treatment with theanti-EGFR activatable antibody being tested). In some embodiments, suchpatients that test negative with respect to a first anti-EGFRactivatable antibody can be tested with other anti-EGFR activatableantibodies comprising different CMs until a suitable anti-EGFRactivatable antibody for treatment is identified (e.g., an anti-EGFRactivatable antibody comprising a CM that is cleaved by the patient atthe site of disease).

Pharmaceutical Compositions

The activatable anti-EGFR antibodies of the invention (also referred toherein as “active compounds”), and derivatives, fragments, analogs andhomologs thereof, can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise theactivatable antibody and a pharmaceutically acceptable carrier. As usedherein, the term “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL′ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1. Materials and Methods

Activatable anti-EGFR antibody expression and purification. The cDNAcoding for the heavy chain and the light chain of each of theactivatable anti-EGFR antibodies were separately cloned into a modifiedpcDNA3.1 mammalian expression vector (Life Technologies). CHO-S cells(Life Technologies) were transiently transfected with the plasmids foreach activatable anti-EGFR antibody for 5-7 days using FreeStyle MAXtransfection reagent (Life Technologies) following the manufacturer'sinstructions. The activatable anti-EGFR antibodies were purified using aHiTrap Mab Select Sure protein A column (GE Healthcare) coupled to anAKTA purifier (GE Healthcare). The purity and the homogeneity ofpurified activatable anti-EGFR antibodies were analyzed by SDS-PAGE inreducing and non-reducing conditions and size exclusion chromatographyusing a Superdex 200, 10/300 GL column (GE Healthcare), respectively.

Rate of activatable anti-EGFR antibody cleavage and Kcat/Kmdetermination. Recombinant human uPA and MT-SP1 (R & D Systems; finalconcentrations of 1.6-100 nM) was incubated with 500 nM (uPA) and 2 μM(MT-SP1) in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, 5 mMcalcium chloride, (TBST) for 24 h at 37° C. The reaction was stopped byadding 5 μl of sample to 7 μl of HT Protein Express Sample Buffer(Caliper LifeSciences) and incubating for 10 min at 95° C. Samples wereanalyzed by capillary electrophoresis (GXII; Caliper LifeSciences) andconcentrations of cleaved and uncleaved light chain were determinedusing LabChip GX software (Caliper LifeSciences). The kcat/Km weredetermined using the following equation:

$\frac{kcat}{Km} = {{- {\ln ( {1 - C} )}}\text{/}( {t^{*}p} )}$

C=relative portion of product converted (cleaved lightchain/cleaved+uncleaved light chain), t=time (s) and p=proteaseconcentration (M). The substrate concentration was maintained below theKm and in excess of the protease.

Protease digestion/activation of activatable anti-EGFR antibody. The3954-1204-C225v4 activatable antibody, also referred to herein asPb-1204, (100 ug) was incubated with various proteases: uPA (50 nM inPBS, pH 7.2), MT-SP1 (10 nM in PBS, pH 7.2), or legumain (30 ug/ml in 50mM MES, 250 mM NaCl, pH 6.0) at room temperature for 4 days. For thecell proliferation and the EGFR binding assays, the uPA-cleaved Pb-1204was purified by protein A agarose affinity chromatography. The cleavageof Pb-1204 was analyzed by SDS PAGE and capillary electrophoresis(LabChip GXII; Caliper Life Sciences).

Cell culture and proliferation assays. H292 human lung cancer cells(American Type Culture Collection) were maintained at 37° C. (5% CO₂) inRPMI medium supplemented with 10% fetal bovine serum (FBS). Forproliferation assays, the cells were seeded at 3000 cells/well in a96-well plate under low serum conditions (RPMI+1% FBS). The followingday, antibodies or activatable anti-EGFR antibodies were added at theindicated concentrations and cells were incubated for an additional 4days at 37° C. Cell viability was measured using Cell-Titer Glo(Promega), according to the manufacturer's instructions.

EGFR binding assay. 96-well plates (Nunc) were coated with EGFR-Fc (50ng/well; R&D Systems) in Hank's Balanced Salt Solution (HBSS pH 7.4, 10mM Hepes) and blocked with HBSS containing 1% BSA. The plates wereincubated with the indicated concentrations of antibody or activatableanti-EGFR antibody in HBSS/1% BSA for 1 h at room temperature. Theplates were then incubated with horseradish peroxidase (HRP) conjugatedanti-human F(ab′)₂ (Jackson ImmunoResearch Laboratories) in HBSS for 30min. and the detection was performed by the addition of3,3′,5,5′-tetramethylbenzidine substrate (1-Step Ultra-TMB, Pierce)followed by an equal volume of 1M hydrochloric acid. Absorbance at 450nm was then measured and reported as optical density (OD 450 nm).

In some cases, the relative EGFR binding was converted to an IgGconcentration using a cetuximab standard curve (1 nM startingconcentration, 10 point serial dilutions). The standard curve was fittedon a 4 parameter curve and the results interpolated using the Elx 800software.

Evaluation of activatable anti-EGFR antibody distribution in vivo byoptical imaging. HT29 xenograft tumor bearing mice were injectedintraperitoneally with 12.5 mg/kg Alexa Fluor 750 conjugated activatableanti-EGFR antibodies. One hour before imaging the mice were injectedintravenously with quenched PEGylated Cy5.5 substrate probes (2 nmol).The mice were imaged 24 h after activatable anti-EGFR antibody injectionusing an IVIS Spectrum/CT imaging system (Caliper LifeSciences). Duringthe procedure, the mice were kept under gaseous anesthesia (5%isofluorane) at 37° C. Imaging at 750 nm is used to evaluateaccumulation levels of the activatable anti-EGFR antibodies (e.g.,3954-1204-C225v4 or 3954-1204-C225v5 as well as the Pb-NSUB construct inwhich the protease substrate sequence from 3954-1204-C225v4 or3954-1204-C225v5, respectively, has been replaced with a sequence thatis not susceptible to protease cleavage), as distribution of the labeledactivatable anti-EGFR antibody constructs at this wavelength is anindication of antibody activation and EGFR receptor binding. Imaging at680 nm is used to evaluate the level of substrate cleavage by monitoringprobe activation kinetics in tumor tissue. Finally, necropsy is used toevaluate biodistribution ex vivo.

Immunofluorescence. Seven days after the activatable anti-EGFR antibodyinjection the mice were sacrificed, and the tissues samples were excisedand cryo-preserved. Samples of HT29 xenograft tumors and liver weresectioned (5 μm) at −20 C. Sections were stained with AlexaFluor488conjugated donkey, anti-human IgG and counterstained with DAPI antifademounting medium. The stained sections were imaged using a fluorescencemicroscope (Olympus IX 81) and an Imaging Software for Life ScienceMicroscopy Cell.

Human IgG ELISA. 96 well maxisorp plates (NUNC) were coated with mouseanti-Human IgG Fc antibody (50 ng/well; Jackson ImmunoResearch) in HBSS(Invitrogen) and blocked with HBSS containing 1% BSA. Samples were addedto the wells and incubated for 1 h at room temperature. The plates werethen incubated with horseradish peroxidase (HRP) conjugated anti-humanF(ab′)₂ (Jackson ImmunoResearch Laboratories) in HBSS for 30 min. andthe detection was performed by the addition of3,3′,5,5′-tetramethylbenzidine substrate (1-Step Ultra-TMB, Pierce)followed by an equal volume of 1M hydrochloric acid. Absorbance at 450nm was then measured and reported as optical density (OD 450 nm). Astandard curve was generated for each plate using cetuximab (1 nMstarting concentration, 10 point serial). The standard curve is fittedon a 4 parameter curve and the results interpolated using the Elx 800software.

Immunoprecipitation and Western Blotting. Tissues samples werehomogenized in lysis buffer (HBSS/2% Triton X-100, Halt proteaseinhibitor cocktail; Thermo Fisher Scientific) using stainless steelbeads containing Bullet Blender homogenization tubes (Next Advance) at aratio of 1 g tumor/tissue per 2 ml lysis buffer. The tissue homogenatewas centrifuged at 3000×g and the supernatant was further centrifuged at20,800×g at 4° C. for 45 minutes. Tissues lysates were immediatelyincubated with 10 μl Goat anti-human IgG Fc Specific magnetic beads(Bangs Laboratories, Inc.), overnight at 4° C. Beads were washed andthen eluted by denaturation in (5 minutes, 95° C.) in LDS Sample Buffer(Invitrogen) containing beta-Mercaptoethanol (Sigma, St. Louis, Mo.).The samples were electrophoresed, transferred to nitrocellulose, blottedusing HRP-conjugated mouse anti-human IgG Fc (Jackson ImmunoResearch).The bands were visualized using the SuperSignal Pico ChemiluminescentSubstrate (Thermo Fisher Scientific) and the ImageQuant LAS 4000 (GEHealthcare).

In vivo efficacy studies. In vivo studies, conducted at JacksonLaboratory were reviewed and approved by the Institutional Animal Careand Use Committee (IACUC). In vivo studies conducted at Oncotest GmbHwhere reviewed approved by the Regierungs-prasidium Freiburg, Germanyand conducted according to the guidelines of the German Animal WelfareAct.

In Vivo Xenograft Studies. In one set of xenograft studies at JacksonLaboratory, 6-8 week old female NU/J (JAX #2019) mice were inoculatedsubcutaneously in the right hind flank with 5×10⁶ NCI-H292 cells (ATCC)suspended 1:1 with Matrigel™ in serum free media. Clinical observations,body weights and digital caliper tumor volume measurements were made 3×weekly once tumors become measurable. Animals were tumor size rankmatched in cohorts (12 mice/group) with average tumor volumes of ˜150mm³-200 mm³ and treatments were started. Animals were treatedintravenously weekly for 4 weeks. Tumors were measured with caliperstwice a week for the duration of the study. Plasma was collected inK₂EDTA from 3 mice per group at 1 h, 8 h, 24 h, 72 h, the bloodcollection was alternated between mice within a cohort. Four mice pergroup were euthanized on Day 3 and tumors were collected and snap frozenfor analysis.

Another set of xenograft studies, using the LXFA677 xenograft model, wasestablished at Oncotest GmbH from primary patient material afterinformed consent. Xenografts were subcutaneously grown in athymic NMRInu/nu mice and randomized after reaching tumor volumes of 100-300 mm³.Mice (12/group) were treated with antibodies once a weekintraperitoneally and tumors were measured with calipers twice weekly.Blood was collected in 3 mice per group once weekly and the bloodcollection was alternated between mice within a cohort. Four mice pergroup were euthanized on Day 3 and tumors were collected and snap frozenfor analysis.

Immunohistochemistry: Samples of xenograft tumors and liver weresectioned (5 μm). Slides were deparaffinized and rehydrated in distilledwater followed by retrieval with Citrate Buffer pH 6.0 (ThermoScientific). Endogenous peroxidase was quenched with 0.3% HydrogenPeroxide. Sections were blocked with the Avidin/Biotin blocking kit(Vector Laboratories) followed by 3% BSA. The sections were stained withBiotin-conjugated donkey, anti-human IgG (Jackson) antibodies utilizingthe ABC Elite Detection kit (Vector Laboratories) and visualized withDAB (Pierce Scientific). The slides were counterstained withHematoxylin, dehydrated, cleared and cover-slipped. The stained sectionswere imaged using a bright field scope (Leica DM750) and LAS EZ software(Leica Application Suite).

Pharmacokinetics: Plasma pharmacokinetic parameters were derived fromcetuximab or activatable anti-EGFR antibody concentrations using anon-compartment analysis with sparse sampling (Phoenix WinNonlin, v 5.2;Pharsight; Mountain View, Calif.) with pooling of individual animalconcentrations for each group at each time point. Values are reported aspopulation estimates with a standard error of the estimate.

Example 2. Preparation of Activatable Anti-EGFR Antibodies

The activatable anti-EGFR antibodies described herein include anantibody or antigen-binding fragment that is derived from the anti-EGFRantibody cetuximab. Cetuximab is a well characterized therapeuticantibody approved for the treatment of colorectal and head and neckcancer. One cetuximab-based activatable anti-EGFR antibody, referred toherein as Pb-1204, was engineered by adding a 21 amino acid long bindingpeptide, i.e., the masking moiety, to the N-terminus of the light chain(FIG. 2A). The mask was attached to the antibody through a 26 amino acidlong linker carrying an 8 residue long sequence that is an uPA, MT-SP1or legumain specific substrate sequence. The efficiency, cleavage rateand selectivity of various proteases (uPA, MT-SP1, tissue plasminogenactivator (tPA) and/or thrombin) with respect to the substrate withinthe Pb-1204 activatable antibody (referred to herein as the 1204substrate or simply 1204) is shown in FIGS. 9A-9D.

The efficiency and the specificity of cleavage of Pb-1204 by differentproteases were evaluated. Pb-1204 was efficiently cleaved by humanMT-SP1 (2200 M-1 s-1±470 M-1 s-1) human uPA (530 M-1 s-1±40 M-1 s-1) andlegumain. However Pb-1204 was not cleaved by tPA or plasmin, ADAM 9,10,and 17, kallikrein 5 and 7. Pb-1204 was digested to near completion andanalyzed by SDS-PAGE, capillary electrophoresis and N-terminalsequencing (FIGS. 2C and 5B). The N-terminal sequence of the cleavedproducts demonstrated the predicted cleavage sites for the threeproteases. MT-SP1 and uPA cleaved the Pb-1204 substrate sequence at thesame P1 site and yielded light chain products of the same molecularweight. The cleavage by legumain yielded a slower migrating light chainthat was shorter by 3 amino acids.

Example 3. In Vitro Activity of Activatable Anti-EGFR Antibodies

The relative in vitro activity of cetuximab, Pb-1204 and Pb-1204digested to completion with uPA was evaluated next. The binding ofPb-1204 to EGFR was reduced by 23-fold relative to cetuximab showing anapparent K_(d) of 0.36 nM compared to 0.02 nM respectively (FIG. 5C).The apparent K_(d) of the fully activated Pb-1204 and cetuximab wereidentical (0.02 nM). The inhibitory activity of Pb-204 on H292 cellproliferation was reduced by 990-fold relative to cetuximab (IC₅₀ 46.6nM and 0.05 nM respectively; FIG. 2D). Once activated, Pb-1204 showed aninhibitory activity comparable to cetuximab (IC₅₀ 0.07 nM).

Example 4. In Vivo Distribution of Activatable Anti-EGFR Antibodies

To assess the in vivo distribution of the proteolytic activitysusceptible of cleaving Pb-1204, near-infrared fluorescenceself-quenched imaging (IQ) probes were used. An IQ probe bearing thesubstrate sequence of Pb-1204 (IQ-1204; FIG. 3A) and a negative controlIQ probe bearing a peptide linker without a substrate sequence (IQ-NSUB)were injected in HT29 tumor bearing mice. The term “NSUB” used hereinrefers to an amino acid substrate sequence that includes glycine andserine residues, but is not susceptible to protease cleavage. One hourafter the injection of IQ-1204, the imaging showed fluorescencerestricted to the tumor site (FIG. 3B). The simultaneous imaging ofthese mice 48 hours after the injection with a fluorescently labeledPb-1204 demonstrated a fluorescence distribution identical to IQ-1204and restricted to the tumor. No fluorescence was detected in the animalsinjected with the IQ-NSUB and fluorescently labeled Pb-NSUB negativecontrols. Seven days after injection, the localization of theactivatable anti-EGFR antibody in tumor and liver samples from treatedmice using immunofluorescence staining was evaluated. Tumors of animalsinjected with Pb-1204 demonstrated membranous fluorescent staining onepithelial cells, whereas no staining was detected in tumors from miceinjected with Pb-NSUB (FIGS. 3C and 3D). No staining was detected inliver samples from animals treated with Pb-1204 or Pb-NSUB (FIGS. 6A and6B). These results indicate that the proteolytic activity associatedwith the tumor site specifically cleaves the 1204 substrate sequence.The cleavage of the substrate sequence allows the antibody to bind toits target and accumulate at the tumor site.

Example 5. Analysis of Activatable Anti-EGFR Antibodies in XenograftModels

The effect of Pb-1204 on EGFR-dependent tumor growth was investigatedusing the NSCLC xenograft model H292. Tumor regression was seen in thePb-1204 treated group and the cetuximab control treated group (FIG. 4A).The mean tumor volume between these treatment groups was notstatistically different at study termination (day 20) suggesting thatPb-1204 was as effective as cetuximab. No statistically significanttumor reduction was seen in mice treated with Pb-NSUB control. In asubset of mice (n=4), plasma samples were collected at different timesup to 72 hours at which point the mice were sacrificed and the tumorswere excised. Despite the circulating concentration of Pb-1204 beinghigher than the cetuximab and Pb-NSUB concentrations, the resulting EGFRbinding activity of Pb-1204 (2.2%±0.2) and Pb-NSUB (3.9%±0.3) wasmarginal compared to cetuximab (95.6%±3.5; FIG. 4B). The concentrationsof Pb-1204, Pb-NSUB and cetuximab found in the tumor were equivalent.The western blot analysis of lysates from these tumors showed that asignificant fraction of the Pb-1204 had been activated, whereas thePb-NSUB remained intact and unactivated. As expected the EGFR bindingactivity of Pb-1204 (51.6%±0.8) found in the tumors was greater than itsactivity in plasma or to the binding activity of Pb-NSUB (18.2%±0.3;FIG. 4C).

Next, the activity of a single dose of Pb-1204 relative to cetuximab wasdetermined in a patient derived non-small cell lung cancer tumorxenograft model (LXFA677). The activatable anti-EGFR antibody and theantibody were equally efficacious at inhibiting the tumor growth. At day21, cetuximab had inhibited tumor growth by 96% and Pb-1204 by 100%(FIG. 4D). The plasma concentrations of cetuximab and Pb-1204 wereequivalent throughout the study (FIG. 7B) with identical exposures (3331μg/ml×day). The concentration EGFR binding activity in plasma wasequivalent to the concentration of the antibody for cetuximab. However,for Pb-1204, the concentration of the EGFR binding activity was greatlydiminished compared to the total concentration of circulatingactivatable anti-EGFR antibody. The exposure (day 1-28) for the EGFRbinding activity of the Pb-1204 (1214 μg/ml×day) represented 36% of theexposure for the total activatable anti-EGFR antibody circulatingconcentration. Seventy two hours after treatment, the tumors from asubset of mice (n=4) were excised and evaluated for activatableanti-EGFR antibody and antibody concentration and activity. The averageconcentration of Pb-1204 in the tumor was equivalent to theconcentration of cetuximab. A considerable fraction (35±4%) of theactivatable anti-EGFR antibody found in the tumors demonstrated EGFRbinding activity.

In summary, through the development of two bacterial display screeningplatforms, an inhibitory binding peptide for cetuximab, i.e., a mask,and a novel peptide substrate for the proteases MT-SP1, uPA and legumainwas identified. The parental antibody was modified to link theinhibitory peptide to the N-terminus of the light chain trough aflexible peptide linker bearing the novel peptide substrate. Thisactivatable anti-EGFR antibody was named Pb-1204. The in vitrocharacterization of Pb-1204 showed that the masking peptide considerablyinhibited the activity of the activatable anti-EGFR antibody compared tocetuximab. The substrate containing linker allows for specificactivation of the activatable anti-EGFR antibody by MT-SP1, uPA andlegumain, and once activated, the activatable anti-EGFR antibodydemonstrated an activity equal to cetuximab. In vivo, Pd-1204 remainedstable in circulation and in normal tissues but was efficientlyactivated in xenograft tumors. The specific tumor activation of Pb-1204led to an anti-tumor efficacy that was equivalent to cetuximab.

Thus, the activatable anti-EGFR antibodies described herein are usefulin improving the therapeutic window of a majority of antibodytherapeutics demonstrating target mediated toxicity. In addition, theuse of the activatable anti-EGFR antibody expands the cancer targetlandscape by allowing the development of cancer therapeutics directedtoward targets that cannot be considered with traditional antibodies dueto associated toxicities.

Example 6. Analysis of Activatable Anti-EGFR Antibodies in Non-HumanPrimate Models

Among the toxicities seen in humans and non-human primates administeredcetuximab is skin rash, including papulopustular rash of face and uppertrunk, and dry and itchy skin. In the studies described herein, theactivatable anti-EGFR antibody 3954-1204-C225v5 was evaluated using anon-human primate animal model. Briefly, three groups of 3 femalecynomolgus monkeys were dosed intravenously (IV) with cetuximab (i.e.,unmodified cetuximab), 3954-1204-C225v5, or Pb-NSUB (i.e.,3954-NSUB-C225v5, an activatable anti-EGFR antibody that includes theNSUB sequence that is not susceptible to protease cleavage),respectively. A 40 mg/kg loading dose was administered (three 2-hourinfusions spread over 5 days), followed by a 25 mg/kg dose that wasadministered weekly (two 2-hour infusions per week). The animals weredosed over a 5-week period (last dose was administered on day 32),followed by a 4-week treatment-free recovery phase. All animals examineddaily for skin rash. All animals were also subject to clinical chemistryand hematology analysis as well as TK and immunogenicity. As shown inFIG. 12 and Table 4, the animals that were administered the activatableanti-EGFR antibody 3954-1204-C225v5 exhibited no detectable rash ascompared to those that were administered cetuximab. In addition, theanimals that were administered the activatable anti-EGFR antibody3954-1204-C225v5 exhibited reduced dermatological toxicity as comparedto those that were administered cetuximab.

TABLE 4 Summary of Dermatological Toxicity Results Observed in Non-HumanPrimates 25 mg/kg Study day Groups Animal No. 22 25 27 29 32 35 40Cetuximab WT0101 1 1 1 2 2 2 2 WT0102 (a) 0 0-1 0 0-1 0-1 0-1 0 WT0103 11 1 2 2 2 1 Pb-NSUB WT1101 0 0 0 0 0 0 0 WT1102 0 0 0 0 0 0 0 WT1103 0 00 0 0 0 0 3954-1204-C225v4 WT2101 0 0 0 0 0 0 0 WT2102 0 0 0 0 0 0 0WT2103 0 0 0 0 0 0 0 0 = No erythema 1 = Slight erythema 2 =Well-defined erythema 3 = Moderate of severe erythema 4 = Severeerythema of slight eschar formation (a) = The “redness” noted on thisanimal was not typical of the “rash” noted in the other two animals.

A comparison of the level of cetuximab and 3954-1204-C225v5 detected inthe blood of the cynomolgus monkeys that received 25 mg/kg weekly dosingafter the initial 40 mg/kg loading dose is shown in FIGS. 13A-13D.

IHC analysis of human IgG in the skin of the cynomolgus monkeysindicated significant binding of cetuximab in skin but no detectablebinding of anti-EGFR activatable antibody 3954-1204-C225v5 in skin.

A pharmacokinetic study comparing serum concentrations over timeindicated that the half-life of anti-EGFR activatable antibody3954-1204-C225v5 was 6.6±0.5 days while the half-life of cetuximab was3.2±0.4 days. An exemplary study showing the half-life of the anti-EGFRactivatable antibody 3954-1204-C225v5 as compared to the half-life ofcetuximab is shown in FIG. 19. The half-life of cetuximab was similar topreviously reported values. The pharmacokinetics of the anti-EGFRactivatable antibody 3954-1204-C225v5 is consistent with lack ofsystemic antigen-mediated clearance (EGFR in normal tissues).

Example 7. Imaging and Immunohistochemistry Staining of Tumor Tissueswith all Active Site Antibodies

The studies described herein were designed to demonstrate that a targetprotease of the activatable anti-EGFR antibodies described herein,specifically the MT-SP1 protease, is active in tumor tissue.

As shown in FIGS. 14A-14C, in vivo imaging and immunohistochemistry(IHC) staining of tumor tissues with A11 active site antibodies (Darraghet al., “Tumor detection by imaging proteolytic activity.” Cancer Res.70 (2010): 1505-1512) confirmed the presence of active MT-SP1 in theH292 xenograft model. All is an antibody that specifically binds to theactive site of the MT-SP1 protease, also known as matriptase.

Example 8. In Situ Imaging of Anti-EGFR Activatable Antibodies

The present Example describes the use of in situ imaging of theactivation and binding of an anti-EGFR activatable antibody of thedisclosure. The results indicate that anti-EGFR activatable antibodiesof the disclosure can be activated by proteases expressed by a tissueand bind EGFR targets on that tissue.

In situ imaging of activatable antibodies represents a unique approachto characterize protease activity in cells and tissues. This technologyenables validation of activatable antibody activation and binding to atarget in histological sections of cells and tissues expressingproteases capable of cleaving the activatable antibody. A schematic ofsuch an in situ approach is presented in FIG. 15.

In situ imaging of the activation and binding of an anti-EGFRactivatable antibody (also referred to herein as in situ imaging) by acell or tissue capable of cleaving the activatable antibody at a siteco-localized with the target recognized by the activated antibody wasconducted as follows: Frozen tissue sections were laid over glassslides. A solution containing labeled anti-EGFR activatable antibodies(labeled, e.g., with a fluorescent tag) was applied on the tissue andincubated, e.g., for 1 hour at room temperature (about 22-24° C.) in anincubation buffer of 50 mM Tris-HCl buffer pH 7.4, containing 150 mMNaCl, 100 μM ZnCl₂, 5 mM CaCl₂) and 0.05% Tween 20; activatable antibodyat a concentration of about 1 μg/ml. The conditions of such anincubation can be adjusted to be conducive to the cleavage agent in thetissue section by, for example, varying the pH of the solution (e.g.,within a range of about pH 7 to about pH 8.5), the temperature of theincubation (e.g., within a range of about 20° C. to about 40° C., e.g.,room temperature or 37° C.), the incubation time (e.g., within a rangeof about 15 minutes to about 150 minutes, and/or the activatableantibody concentrations (e.g., within a range of about 0.05 μg/ml toabout 10 μg/ml). The tissue was then extensively washed to removenon-bound material and detectable label was measured. For example, whena fluorescent tag was used, the tissue was submitted to fluorescentmicroscopy. Detection of activated antibody on the tissue indicated thatthe tissue expressed proteases that cleaved the activatable antibody andalso expressed EGFR targets to which the activated antibody bound.

The ability of anti-EGFR activatable antibody 3954-1204-C225v5 to beactivated and to bind frozen human lung cancer tissue was evaluatedusing in situ imaging. The activatable antibody was labeled with AlexaFluor® 680 (Invitrogen) to produce labeled activatable antibody3954-1204-C225v5-AF680. The labeled activatable antibody was incubatedwith a frozen human lung cancer tissue sample as described above. Theresults are shown in FIG. 16, panel A. The red fluorescent tissue imagedemonstrates binding of C225v5 antibody activated by tissue-derivedproteolytic cleavage of the anti-EGFR activatable antibody. Theidentical pattern of tissue staining was detected by exposing an EGFRantibody (monoclonal rabbit anti-EGFR antibody, Cell Signaling) to thetissue, as shown in FIG. 16, panel B. The fluorescent signal shown inpanel A was inhibited by pre-treatment of the tissue with a 1:100dilution of broad spectrum inhibitor cocktail set III (539134, EMDMillipore, Billerica, Mass.) and 50 mM EDTA, as shown in FIG. 16, panelC. Blue staining represents DAPI nuclear staining.

FIG. 17 demonstrates that 3954-1204-C225v5 is activatable in a widerange of human tumor samples. Column 2 indicates the expression level ofEGFR receptor, as detected by an EGFR antibody (monoclonal rabbitanti-EGFR antibody, Cell Signaling), for the various human cancer tissuesamples. Column 3 indicates the amount of active matriptase (MT-SP1), asdetected by antibody A11, in the various human cancer tissue samples.Columns 4 and 5 represent an evaluation of in situ activation andbinding of the EGFR activatable antibody (col. 5) as compared tocetuximab (Cetux) tissue staining (col. 4). The staining that measuresthe amount of EGFR, A11 and cetuximab antibodies binding to the tissuesample was scored from 0 to 3+: 0, no staining; 1+ (i.e., “+”), weakstaining; 2+ (i.e., “++”), moderate staining; and 3+ (i.e., “+++”),strong staining. The activatable antibody in situ imaging stainingscoring is based on comparison with cetuximab antibody staining anddefined as follows: 0, no staining; 1+ (i.e., “+”), weak staining ascompared to parental antibody; 2+ (i.e., “++”), moderate staining ascompared to parental antibody; and 3+ (i.e., “+++”), analogous stainingto parental antibody. As shown in FIG. 17, high levels of activematriptase have been observed in 8 of 9 samples from colorectal cancer(CRC) tumors, and high levels of active matriptase have been observed insamples from 5 of 10 lung cancer (NSCLC) tumors. No active matriptasewas observed in samples from adjacent healthy lung tissue.

These data suggest the utility of in situ imaging in methods ofeffectively and efficiently identifying or otherwise refining a patientpopulation suitable for treatment with an anti-EGFR activatable antibodyof the disclosure, such as activatable antibody 3954-1204-C225v5. Forexample, patients that test positive for both the target (e.g., EGFR)and the protease that cleaves the substrate in the cleavable moiety (CM)of the anti-EGFR activatable antibody (e.g., MT-SP1) using these in situimaging techniques could be identified as suitable candidates fortreatment with the anti-EGFR activatable antibody being tested.Likewise, patients that test negative for either or both of the target(e.g., EGFR) and the protease that cleaves the substrate in the CM(e.g., MT-SP1) using these in situ imaging techniques are identified assuitable candidates for another form of therapy (i.e., not suitable fortreatment with the anti-EGFR activatable antibody being tested). In someembodiments, such patients can be tested with other anti-EGFRactivatable antibodies until a suitable anti-EGFR activatable antibodyfor treatment is identified (e.g., an anti-EGFR activatable antibodycomprising a CM that is cleaved by the patient at the site of disease).

Example 9. Activatable Antibodies Conjugated to an Agent

The present Example demonstrates the ability of an activatableantibody-agent conjugate of the disclosure to inhibit tumor growth.

Activatable antibody 3954-1204-C225v5 was conjugated to monomethylauristatin E (MMAE), a synthetic anti-mitotic tubulin polymerizationinhibitor via a Val-Cit-based linker (K-lock C5-vc-PAB; ConcortisBiosystems Corp., San Diego, Calif.), to generate activatableantibody-agent conjugate 3954-1204-C225v5-MMAE. The in vivo efficacy of3954-1204-C225v5-MMAE relative to 3954-1204-C225v5 and to cetuximab wastested using the H292 lung cancer cell line xenograft mouse model. TheH292 xenograft model was established by inoculating 6-8 week old femaleNU/J (JAX #2019) mice subcutaneously in the right hind flank with 5×10⁶NCI-H292 cells (ATCC) suspended 1:1 with Matrigel™ in serum-free media.Tumor volume measurements were made 3 times (3×) weekly once tumorsbecame measurable. Animals were tumor-size rank-matched in cohorts (8mice/group) with average tumor volumes of ˜150 mm³-200 mm³, andtreatments were administered to the groups 1 time on day 0 as follows:(a) 12.5 mg/kg 3954-1204-C225v5-MMAE, applied intravenously; (b) 2.5mg/kg 3954-1204-C225v5-MMAE, applied intravenously; (c) 12.5 mg/kgrituxan-MMAE (i.e., rituximab-MMAE; rituximab is available fromGenentech, So. San Francisco, Calif.), applied intravenously; (d) 12.5mg/kg 3954-1204-C225v5, applied intraperitoneally; (e) 12.5 mg/kgcetuximab, applied intraperitoneally; or (f) 12.5 mg/kg IVIg, appliedintraperitoneally. Tumors were measured with calipers twice weekly forthe duration of the study.

As shown in FIG. 18, a single injection of 12.5 mg/kg activatableantibody-agent conjugate 3954-1204-C225v5-MMAE demonstrated significantefficacy compared to 12.5 mg/kg IVIg (92% tumor reduction; p=0.0000003),as did a single injection of 12.5 mg/kg cetuximab (78% tumor reduction;p=0.000006) 28 days after administration. The efficacy of3954-1204-C225v5-MMAE was significantly higher than that of cetuximab atthis dose (p=0.03) at day 28. A reduced efficacy was recorded for alower dose (2.5 mg/kg) of 3954-1204-C225v5-MMAE (p=0.007) and as well asfor a dose of 12.5 mg/kg rituxan-MMAE (p=0.008) at day 28. Nostatistically significant efficacy was observed for activatable antibody3954-1204-C225v5 at 12.5 mg/kg (p=0.06 at day 28).

An additional dose fractionation study in the same H292 xenograft tumormodel was run to compare the activity of cetuximab, activatable antibody3954-1204-C225v5-MMAE administered at a single dose of 12.5 mg/kg oradministered twice weekly (at Days 0, 3, 7, 10 and 14) doses of 2.5mg/kg each for a total of 5 doses (total of 12.5 mg/kg activatableantibody administered over the study). This dose fractionation studyused the same control (12.5 mg/kg IVIg) and methods described above forthe H292 xenograft model, EGFR binding assay, and cell culture andproliferation assays. In this dose fractionation study, the activatableantibody 3954-1204-C225v5-MMAE was well tolerated, as evidenced by thelack of weight loss in treated mice. A single control animal waseuthanized on Day 23 with body weight loss >20%, but all other animalssurvived through Day 35.

Significant anti-tumor activity was seen in all activatable antibody3954-1204-C225v5-MMAE and cetuximab treated groups as shown in Table 5below, where values greater than 100% represent tumor shrinkage.

TABLE 5 Tumor growth inhibition relative to control-treated group. TumorGrowth Inhibition (%) Treatment Day 7 Day 14 Day 21 Day 28 Day 35 IVIG25 mg/kg  0%  0%  0%  0%  0% Cetuximab  91% 102%  90%  74%  61% 12.5mg/kg Cetuximab  82%  98%  93%  87%  77% 2.5 mg/kg × 5 3954-1204-C225v5-104% 124% 112% 112% 109% MMAE 12.5 mg/kg 3954-1204-C225v5-  39% 107%107% 107% 107% MMAE 2.5 mg/kg × 5

Cetuximab as a single dose of 12.5 mg/kg produced tumor stasis or tumorregression in 8/8 mice; however, the effect was short lived with 6/8mice showing evidence of tumor regrowth on Day 35. Cetuximab as afractionated dose of 2.5 mg/kg likewise produced tumor stasis orregression in all animals followed by regrowth in 5/8 mice by Day 35.Administration of the activatable antibody 3954-1204-C225v5-MMAE as asingle dose of 12.5 mg/kg had a more pronounced anti-tumor effect,resulting in tumor regressions in 7/7 mice, with no evidence of tumorregrowth by Day 35. Twice weekly administration of 2.5 mg/kg3954-1204-C225v5-MMAE yielded tumor regressions in 8/8 mice, only one ofwhich showed evidence of tumor regrowth on Day 35. Statistical analysisof the Day 35 tumor volumes showed that both regimens using3954-1204-C225v5-MMAE were significantly more efficacious than thefractionated dose of cetuximab (one way ANOVA p<0.005 in each case). Thesame was true when comparing to the cetuximab single dose group. Therewas no significant difference on Day 35 between the activities of thetwo regimens using 3954-1204-C225v5-MMAE (p=0.22 using a two-tailedt-test).

In conclusion, the activatable anti-EGFR antibody-agent conjugate3954-1204-C225v5-MMAE showed robust and durable efficacy in the H292tumor model, with activity superior to a matching dose of cetuximab. Theactivatable antibody-agent conjugate 3954-1204-C225v5-MMAE was equallyeffective when administered as a single dose of 12.5 mg/kg or a repeateddose of 2.5 mg/kg given twice weekly for a total of 5 doses.

Example 10. In Vitro Characterization of an Activatable AntibodyConjugated to an Agent

This Example describes the ability of an activatable antibody-agentconjugate of the disclosure to retain binding to EGFR and inhibitproliferation of H292 cells in culture.

Anti-EGFR activatable antibody 3954-1204-C225v5 was conjugated tomonomethylauristatin E (MMAE), a synthetic anti-mitotic tubulinpolymerization inhibitor as described in the Examples herein, togenerate activatable antibody-agent conjugate 3954-1204-C225v5-MMAE. Theaverage drug to antibody ratio (DAR) was 3.44.

The abilities of the following compounds to bind EGFR in an ELISA-basedassay and to inhibit H292 cell proliferation in cell culture weredetermined: Anti-EGFR activatable antibody-agent conjugate3954-1204-C225v5-MMAE; anti-EGFR activatable antibody-agent conjugate3954-1204-C225v5-MMAE activated by uPA; anti-EGFR activatable antibody3954-1204-C225v5; anti-EGFR activatable antibody 3954-1204-C225v5activated by uPA; and anti-EGFR antibody C225v5. Activation ofactivatable antibody and activatable antibody-agent conjugate waseffected by digestion overnight at 37° C. with active site-titrated uPA(500 nM) in Tris pH 8.5; activation was measured by CE analysis (LabChipGXII). uPA-activated activatable antibody and uPA-activated activatableantibody-agent conjugate were purified using protein A and then storedat 4° C. prior to the study.

For the ELISA-based binding experiments, EGFR-Fc (R&D Systems) wascoated to the wells of a 96-well ELISA plate. A 1:3 serial dilution ofthe compounds was applied to the plate and allowed to bind. Boundantibody was visualized with an anti-human F(ab′)₂ IgG-HRP conjugate(Jackson ImmunoResearch Laboratories) and developed with the chromogenicsubstrate TMB. FIG. 20A demonstrates that conjugation of MMAE to theactivatable antibody did not interfere with the ability of the maskingmoiety to inhibit binding of the activatable antibody to EGFR. Inaddition, conjugation of MMAE to the uPA-activated activatable antibodydid not alter the activated antibody's ability to bind to EGFR.

For the cell proliferation assay, the human lung cancer cell line H292was obtained from ATCC. H292 cells were grown in complete media(RPMI-1640 supplemented with 10% fetal bovine serum) at 37° C. in anatmosphere of 5% CO₂ in air. H292 cells were harvested during thelogarithmic growth period, resuspended in complete medium, and plated ata density of 10,000 cells per well in a 96-well white wall chimneyplate. Following overnight incubation, a 10-point 1:3 serial dilution,starting at 10 μg/ml and ending in essentially 0 μg/ml of each compoundwas added to cells in culture in replicates. Cells were cultured for 3days and cell viability was measured using CellTiterGlo (Promega)following manufacturer's protocol and a luminometer (Tecan). Data wereanalyzed using Prism GraphPad. FIG. 20B demonstrates that conjugation ofMMAE to the uPA-activated activatable antibody increases the activatedantibody's cell-killing activity. In addition, conjugation of MMAE tothe activatable antibody did not interfere with the ability of themasking moiety to inhibit cell killing by the uncleaved activatableantibody.

Example 11. In Situ Imaging of Anti-EGFR Activatable Antibodies

The present Example describes the use of an in situ imaging approach toscreen a patient's tissue samples for the activation and binding of ananti-EGFR activatable antibody. The results indicate that anti-EGFRactivatable antibodies of the disclosure can be activated by proteasesexpressed by a cancer patient's tissue and bind to EGFR receptor on thattissue.

Human colorectal cancer, non-small cell lung cancer (NSCLC) and livermetastasis tissue samples were profiled for EGFR and MT-SP1 expressionby treating frozen tissue with labeled EGFR and A11 antibodies at 1μg/ml and 5 μg/ml concentrations, respectively for 1 hour (FIG. 21 andTables 6-8). Furthermore, the ability of anti-EGFR activatable antibody3954-1204-C225V5 to be activated and to bind human tumor or livermetastasis tissues was evaluated using in situ imaging. The activatableantibody was labeled with Alexa Fluor® 680 (Invitrogen) as describedabove. The resultant activatable antibody 3954-1204-C225V5-AF680 (alsoreferred to as 1204-C225v5 or 1204-C225) was incubated with frozenpatient tissue samples according to the protocol of in situ imagingdescribed herein. FIG. 22 illustrates the ability of colorectal cancerliver metastasis tissues to activate and bind anti-EGFR activatableantibodies. The results on the ability of cancer patient's tissuesamples to activate and bind anti-EGFR activatable antibodies aresummarized in the Tables 6-8. The IHC staining that measures the amountof EGFR and A11 antibodies binding to the tissue sample was scored from0 to 3+: 0, no staining; 1+ (i.e., “+”), weak staining; 2+ (i.e., “++”),moderate staining; and 3+ (i.e., “+++”), strong staining. The in situimaging staining scoring is based on comparison with cetuximab antibodystaining and defined as follows: 0, no staining; 1+ (i.e., “+”), weakstaining as compared to parental antibody; 2+ (i.e., “++”), moderatestaining as compared to parental antibody; and 3+ (i.e., “+++”),analogous staining to parental antibody.

TABLE 6 Screening for EGFR and MT-SP1 expression and in situ imaging of3954-1204-C225 activatable antibody in colorectal cancer patients tumortissues. IHZ** IHC* Cetux- Activatable AJCC 7th Patient # EGFR MT-SP1imab Antibody Tumor type Stage Grade edition Treatment 5577 +++ +++ ++++ Adenocarcinoma N/A G2 Moderately N/A UNKNOWN Differentiated 5579 +++− ++ ++ Adenocarcinoma N/A G3 Poorly (pT4, pN2, UNKNOWN DifferentiatedpM1) 5638 ++ + ++ ++ Adenocarcinoma Stage IIA G2 Moderately (pT3, pN0,UNKNOWN Differentiated pMn/a) 5640 ++ ++ ++ +++ Adenocarcinoma Stage IIAG2 Moderately IIA (pT3, pN0, UNKNOWN Differentiated pMn/a) 5642 ++ ++++ + Adenocarcinoma Stage IIA G3 Poorly II (pT3, pN0, UNKNOWNDifferentiated pMn/a) 5650 ++ + ++ ++ Adenocarcinoma Stage IIIB G2Moderately IIIB (pT3, UNKNOWN Differentiated pN0, pMn/a) 5652 +++ ++++++ ++ Adenocarcinoma Stage IIA G2 Moderately (pT3, pN0, UNKNOWNDifferentiated pMn/a) 5656 +++ ++ +++ +++ Adenocarcinoma Stage IIB G2Moderately (pT3c/d, pN1, UNKNOWN Differentiated pMn/a) 5658 + ++ ++ +++Adenocarcinoma Stage IIIB G3 Poorly (pT3, pN1, UNKNOWN DifferentiatedpMn/a) 5660 +++ +++ ++ +++ Adenocarcinoma Stage I G2 Moderately (pT2,pN0, UNKNOWN Differentiated pMn/a) 5662 − − − − Adenocarcinoma Stage IIAG2 Moderately (pT3, pN0, UNKNOWN Differentiated pMn/a) 5663 + − + −Adenocarcinoma Stage IIA G3 Poorly (pT3, pN0, UNKNOWN DifferentiatedpMn/a) 5665 ++ + ++ ++ Adenocarcinoma N/A N/A (pT4, pN1, UNKNOWN pMn/a)*The IHC staining scored from 0 to 3+ that measures the amount ofantibody binding: 0, no staining; 1+, weak staining; 2+, moderatestaining; and 3+, strong staining. **The IHZ staining scoring is basedon comparison with parental antibody staining: 0, no staining; 1+, weakstaining as compared to parental antibody; 2+, moderate staining ascompared to parental antibody; and 3+, analogous staining to parentalantibody.

TABLE 7 Screening for EGFR and MT-SP1 expression and in situ imaging of3954-1204-C225 activatable antibody in human colorectal cancer livermetastasis tissues. IHZ** Activatable IHC* Antibody w/ Tumor AJCC 7thPatient # EGFR MT-SP1 Cetuximab 1204 type Stage Grade edition Treatment10398 ++ ++ ++ + Metastatic N/A N/A N/A N/A Adenocarcinoma 10404 +++ +++++ +++ Metastatic Stage IV N/A N/A Yes-N/A Adenocarcinoma 10444 +++ +++ +++ Metastatic Stage IV N/A N/A Yes-N/A Adenocarcinoma 10465 ++ ++ ++++ Metastatic Stage IV N/A N/A Yes-N/A Adenocarcinoma 10470 ++ ++ ++ ++Metastatic Stage IV N/A N/A FOLFIRI Adenocarcinoma Avastin 10484 − ++ −− Metastatic Stage IV N/A N/A FOLFOX Adenocarcinoma 10498 ++/+ ++ ++ +++Metastatic N/A N/A N/A Chemo- Adenocarcinoma FOLFIRI Radiation-PelvicXRT 10510 ++ +++ + +++ Metastatic Stage IV N/A N/A Chemo-5FUAdenocarcinoma Leucovorin Radiation-Pelvic XRT 10515 + ++ + +/−Metastatic N/A N/A N/A FOLFIRI Adenocarcinoma Avastin 10517 ++ + ++/++++/++ Metastatic Stage IV N/A N/A FOLFOX Adenocarcinoma 10519 ++ + +++++ Metastatic G2, N/A Folfox Adenocarcinoma Moderately (Previous)Differentiated CPT11, Leucovorin, 5FU (Current) *The IHC staining scoredfrom 0 to 3+ that measures the amount of antibody binding: 0, nostaining; 1+, weak staining; 2+, moderate staining; and 3+, strongstaining. **The IHZ staining scoring is based on comparison withparental antibody staining: 0, no staining; 1+, weak staining ascompared to parental antibody; 2+, moderate staining as compared toparental antibody; and 3+, analogous staining to parental antibody.

TABLE 8 Screening for EGFR and MT-SP1 expression and in situ imaging of3954-1204-C225 activatable antibody in NSCLC patients tissues. IHZ**Activat- IHC* able AJCC 7th Patient # EGFR MT-SP1 Cetuximab AntibodyTumor type Stage Grade edition Treatment 5648 − − − − ADENO- Stage I G2Moderately IB (pT2, pN0 UN- CARCINOMA differentiated (0/12), pM n/a)KNOWN 5608 − +++ − − ADENO- Stage I G2 Moderately IB (pT2, NONECARCINOMA differentiated pN0(0/12), pM n/a) 5624 − − − − Squamous CellStage I G2 Moderately IB (pT2a, pN0, NONE Carcinoma differentiatedpMn/a) 5613 + − ++ + squamous cell Stage I G3 Poorly IB (pT2a, pN0, NONEcarcinoma differentiated M n/a). 5614 +++ ++ +++ ++ Squamous Cell StageI G3 Poorly IB (pT2, NONE Carcinoma differentiated pN0(0/12), pM n/a)5633 + +/− + + ADENO- Stage I G2 Moderately IA (pT1b, pN0, YES-N/ACARCINOMA differentiated pMnia) 5636 + + + + Large Cell Stage I G3Poorly IA (pT1a, pN0, NONE Carcinoma differentiated pMn/a) 5630 + + + −Squamous Cell Stage I G2 Moderately IB (pT2a, pN0, NONE Carcinomadifferentiated pMn/a) 5618 +++ + +++ +++ Squamous Cell Stage I G2Moderately IB (pT2, pN0, NONE Carcinoma differentiated pMn/a) 5619 + +/−++ + ADENO- Stage II G2 Moderately IIB (pT2,pN1, NONE CARCINOMAdifferentiated pM n/a) 5627 ++ − ++ Squamous Cell Stage II G2 ModeratelyIIB (pT2b, pN1, NONE Carcinoma differentiated pM n/a) 5629 +++ − +++ ++ADENO- Stage II G2 Moderately IIB (pT2b, pN1, NONE CARCINOMAdifferentiated pMn/a) 5646 +/− + + + LARGE CELL Stage II G3 Poorly IIB(pT2, pN1, UN- NEURO- differentiated pMX). KNOWN ENDOCRINE CARCINOMA5654 +/− ++ − − LARGE CELL Stage II G3 poorly IIA (pT2, pN0, UN- NEURO-differentiated pMn/a) KNOWN ENDOCRINE CARCINOMA 5607 − + − − ADENO-Stage III G3 Poorly IIIA (pT2, pN0, NONE CARCINOMA differentiated pMn/a)5610 − − − − Squamous Cell Stage III G2 Moderately IIIA (pT3, pN1) NONECarcinoma differentiated 5615 + − ++ + squamous cell Stage III G3 PoorlyIIIA (pT3, pN1, NONE carcinoma differentiated M n/a) 5620 +/− − − −Large Cell Stage III G3 Poorly IIIA (pT3, pN1, NONE Basaloiddifferentiated pMn/a) Carcinoma 5621 +++ ++ +++ ++ SQUAMOUS Stage III G2Moderately IIIA (pT4, pN0, NONE CELL differentiated M n/a). CARCINOMA5625 +++ ++ +++ ++ Squamous Cell Stage III G2 Moderately IIIA (pT4, pN2,YES-N/A Carcinoma differentiated pM n/a) *The IHC staining scored from 0to 3+ that measures the amount of antibody binding: 0, no staining; 1+,weak staining; 2+, moderate staining; and 3+, strong staining. **The IHZstaining scoring is based on comparison with parental antibody staining:0, no staining; 1+, weak staining as compared to parental antibody; 2+,moderate staining as compared to parental antibody; and 3+, analogousstaining to parental antibody.

Example 12. Quantification of In Situ Imaging of Anti-EGFR ActivatableAntibodies

The present Example describes in situ imaging of the activation andbinding of an anti-EGFR activatable antibody to biological tissues exvivo in combination with anti-EGFR antibody IHC and A11 antibody IHC.The use of commercially available anti-EGFR antibody IHC allows one tonormalize the staining of parental antibody (e.g. cetuximab) andanti-EGFR activatable antibody to the quantity of EGFR expression in atissue. Co-staining with EGFR antibodies also enables qualitativequantification of in situ imaging of anti-EGFR activatable antibodiesrelative to the cetuximab staining normalized to EGFR expression.Quantification of the fluorescent signal can be performed usingbioanalytical software for research imaging, such as MetaMorph. Thestaining of tissue with antibody that specifically recognizes the activesite of MT-SP1 (antibody A11) can also be performed to monitor theactivity of MT-SP1, an enzyme that proteolytically cleaves the substrateof activatable antibodies 3954-1204-C225v4, 3954-1204-C225v5, and3954-1204-C225v6.

Quantification of in situ imaging of anti-EGFR activatable antibodycleavage by a cell or tissue performed in combination with EGFR IHC wasconducted as follows: Frozen tissue sections were laid over glass slidesand rinsed in 1×PBS-T. A solution containing labeled anti-EGFRactivatable antibodies (labeled, e.g., with a fluorescent tag) wasapplied on the tissue and incubated, e.g., for 1 hour at roomtemperature in an incubation buffer of 50 mM Tris-HCl buffer pH 7.4,containing 150 mM NaCl, 100 μM ZnCl₂, 5 mM CaCl₂) and 0.05% Tween 20;activatable antibody at a concentration of about 1 μg/ml. The tissue wasthen rinsed in 1×PBS-T to remove non-bound material, and endogenous IgGwas blocked with 3% BSA. Sections were incubated with commerciallyavailable anti-Rabbit, anti-EGFR antibodies and labeled A11 antibodies(labeled, e.g., with a fluorescent tag), e.g., for 1 hour at roomtemperature. After rinsing, secondary antibody Anti-Rabbit IgG labeledwith a fluorescent tag was applied and incubated on the sections e.g.,for 30 minutes at room temperature at a concentration of 5 μg/ml toamplify the primary antibody. Sections were rinsed and detectable labelwas measured. For example, when a fluorescent tag was used, the tissuewas submitted to fluorescent microscopy. The ability of anti-EGFRactivatable antibody to be activated and to bind to the receptor in situwas quantified by the following equation:

${PbA} = {{\frac{( {{Ab}\mspace{11mu} {{EGFR} \cdot {Pb}}\mspace{14mu} {in}\mspace{14mu} {situ}\mspace{14mu} {imaging}} )}{( {{Pb}\mspace{11mu} {{EGFR} \cdot {Ab}}\mspace{14mu} {in}\mspace{14mu} {situ}\mspace{14mu} {imaging}} )} \cdot 100}\%}$

PbA=% of anti-EGFR activatable antibody activation and binding ascompared to parental antibody (e.g. cetuximab), Ab EGFR=stainingintensity of EGFR IHC on the section with cetuximab binding, PbEGFR=staining intensity of EGFR IHC on the section with anti-EGFRactivatable antibody in situ imaging, Ab in situ imaging=intensity ofcetuximab binding, Pb in situ imaging=intensity of anti-EGFR activatableantibody binding.

Human esophageal and pancreatic cancer tissue samples were profiled forEGFR and MT-SP1 expression; the ability of anti-EGFR activatableantibody 3954-1204-C225v5 to be activated and to bind human tumor wasevaluated using in situ imaging. The activatable antibody was labeledwith Alexa Fluor® 680 (Invitrogen) as described above. The resultantactivatable antibody 3954-1204-C225v5-AF680 was incubated with frozenpatient tissue samples according to the protocol of in situ imagingdescribed herein. Furthermore, EGFR and Alexa Fluor® 750 labeled A11antibodies were used by treating frozen tissue with at 1 μg/ml and 5μg/ml concentrations, respectively for 1 hour. FIG. 23 illustrates theability of esophageal cancer tissues to activate and bind anti-EGFRactivatable antibodies. The results on the ability of esophageal andpancreatic cancer patients' tissue samples to activate and bindanti-EGFR activatable antibodies are summarized in Table 9. The IHCstaining that measures the amount of Cetuximab, EGFR and A11 antibodiesbinding to the tissue sample was scored from 0 to 3+: 0, no staining; 1+(i.e. “+”) weak staining; 2+ (i.e. “++”) moderate staining; and 3+ (i.e.“+++”) strong staining. The in situ imaging of anti-EGFR activatableantibodies staining was quantified based on comparison with cetuximabantibody staining normalized to EGFR staining and calculated by theequation described above.

TABLE 9 Screening for EGFR and MT-SP1 expression and in situ imaging of3954-1204-C225 activatable antibody in esophageal and pancreatic cancerpatients tumor tissues. IHC in situ imaging Patient MT- 3954-1204- #EGFR SP1 Cetuximab C225 Disease Diagnosis  5586 ++ ++ ++  ~55%Esophageal cancer  5594 +++ ++ +++  ~90% Esophageal cancer  5595 ++ +++++  ~80% Esophageal cancer  5606 +++ +++ +++ ~100% Esophageal cancer 5641 +++ ++ ++  ~90% Esophageal cancer  5587 ++ +++ ++ ~100% Pancreaticcancer  5617 + + +  ~80% Pancreatic cancer  5623 ++ ++ ++  ~75%Pancreatic cancer 13007 ++ ++ ++ ~100% Pancreatic cancer 13011 − + − −Pancreatic cancer *The IHC and Cetuximab staining scored from 0 to 3+that measures the amount of antibody binding: 0, no staining; 1+, weakstaining; 2+, moderate staining; and 3+, strong staining. **The3954-1204-C225 in situ imaging scoring is based on comparison withparental antibody staining normalized to the EGFR IHC staining andidentified as % of activation: 0, no activation; 100% activationresulting in staining analogous to staining to parental antibody.

FIG. 23 is a series of photographs showing the triple staining of insitu imaging, EGFR IHC and A11 IHC. The upper row of images demonstratesthe staining performed on a single tissue slice, demonstrating (left toright): EGFR expression, activity of matriptase (MT-SP1) and binding ofcetuximab under in situ imaging conditions. The lower row of imagesdemonstrates the staining performed on a single tissue slice,demonstrating (left to right): EGFR expression, activity of matriptase(MT-SP1) and in situ imaging of anti-EGFR activatable antibody3954-1204-C225v5. The right column of images in FIG. 23 compares bindingof cetuximab (upper image) and of anti-EGFR activatable antibodyactivated by tissue-derived proteolytic cleavage (lower image) under insitu imaging conditions. The identical pattern of tissue staining wasdetected by exposing a commercially available anti-EGFR antibody to thetissue, as shown in FIG. 23, left column of images. FIG. 23, middlecolumn of images, demonstrates co-localization of matriptase (MT-SP1)activity with EGFR expression. As used herein, the term“co-localization” is not intended to imply any overlay or other overlapof the EGFR and/or A11 staining, and the term “co-localization” is usedto indicate the presence of MT-SP1 activity in EGFR-expressing patienttissue. Overall, these data demonstrate about 90% activation ofanti-EGFR antibody 3954-1204-C255v5 by the human esophageal cancertissue sample.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following.

1-70. (canceled)
 71. A method of treating or delaying the progression ofa cancer associated with Epidermal Growth Factor Receptor (EGFR) in asubject comprising administering to a subject in need thereof atherapeutically effective amount of an activatable antibody that in anactivated state binds EGFR, wherein said activatable antibody comprises:(a) an antibody or an antigen binding fragment thereof (AB) thatspecifically binds to EGFR, wherein the AB comprises: (i) heavy chaincomplementarity determining regions (HCDRs), wherein the HCDRs are theHCDRs of a heavy chain amino acid sequence selected from the groupconsisting of: SEQ ID NO: 26, SEQ ID NO: 30, and SEQ ID NO: 34, (ii)light chain complementarity determining regions (LCDRs), wherein theLCDRs are the LCDRs of light chain amino acid sequence SEQ ID NO: 68,and (iii) a heavy chain comprising an amino acid sequence with at leastone of the following amino acid sequence features: (1) a glutamineresidue at the position corresponding to position 88 of SEQ ID NO: 26,SEQ ID NO: 30, and SEQ ID NO: 34, and (2) an alanine residue at theposition corresponding to position 299 of SEQ ID NO: 26, SEQ ID NO: 30,and SEQ ID NO: 34; (b) a masking moiety (MM) coupled to the AB thatinhibits the binding of the AB of the activatable antibody in anuncleaved state to EGFR; and (c) a cleavable moiety (CM) coupled to theAB, wherein the CM is a polypeptide that functions as a substrate for aprotease, wherein the activatable antibody in the uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-CM-AB or AB-CM-MM.
 72. The method of claim 71, wherein the cancer isbreast cancer, colorectal cancer, gastric cancer, glioblastoma, head andneck cancer, lung cancer, ovarian cancer, endometrial cancer, pancreaticcancer, prostate cancer, renal cancer, sarcoma, skin cancer,triple-negative breast cancer, esophageal cancer, non-small cell lungcancer, osteosarcoma, squamous cell cancer, basal cell carcinoma, ormelanoma.
 73. The method of claim 71, the AB is conjugated to an agent.74. The method of claim 73, wherein the agent has one or more of thecharacteristics selected from the group consisting of: (a) the agent isa toxin or fragment thereof, (b) the agent is a dolastatin or aderivative thereof, (c) the agent is an auristatin or derivativethereof, (d) the agent is auristatin E or a derivative thereof, (e) theagent is monomethyl auristatin E (MMAE), (f) the agent is a maytansinoidor a derivative thereof, (g) the agent is DM1, (h) the agent is DM4, (i)the agent is a duocarmycin or derivative thereof, (j) the agent is acalicheamicin or derivative thereof, (k) the agent is a detectablemoiety, and (l) the agent is a diagnostic agent.
 75. The method of claim71, wherein the AB thereof is selected from the group consisting of aFab fragment, a F(ab′)₂ fragment, a scFv, and a scAb.
 76. The method ofclaim 71, wherein the HCDRs of the AB comprise the amino acid sequencesNYGVH (SEQ ID NO: 71), VIWSGGNTDYNTPFTS (SEQ ID NO: 72), and ALTYYDYEFAY(SEQ ID NO: 73); and the LCDRs of the AB comprise the amino acidsequences RASQSIGTNIH (SEQ ID NO: 74), YASESIS (SEQ ID NO: 75), andQQNNNWPTT (SEQ ID NO: 76).
 77. The method of claim 71, wherein the ABcomprises: (i) a heavy chain variable region, wherein the heavy chainvariable region is the heavy chain variable region of a heavy chainamino acid sequence selected from the group consisting of SEQ ID NO: 26and SEQ ID NO: 34, (ii) a light chain variable region, wherein the lightchain variable region is the light chain variable region of light chainamino acid sequence SEQ ID NO:
 68. 78. The method of claim 71, whereinthe MM has one or more of the characteristics selected from the groupconsisting of: (i) the MM has an equilibrium dissociation constant forbinding to the AB which is greater than the equilibrium dissociationconstant of the AB to EGFR, (ii) the MM does not interfere or competewith the AB of the activatable antibody in a cleaved state for bindingto EGFR, (iii) the MM is a polypeptide of no more than 40 amino acids inlength, (iv) the MM polypeptide sequence is different from that of EGFR,(v) the MM polypeptide sequence is no more than 50% identical to anynatural binding partner of the AB, (vi) the MM does not comprise morethan 25% amino acid sequence identity to EGFR, and (vii) the MM does notcomprise more than 10% amino acid sequence identity to EGFR.
 79. Themethod of claim 71, wherein the wherein the MM comprises the amino acidsequence CISPRGCPDGPYVMY (SEQ ID NO: 14).
 80. The method of claim 71,wherein the CM is a polypeptide of up to 15 amino acids in length orwherein the protease cleaves the CM in the activatable antibody when theactivatable antibody is exposed to the protease.
 81. The method of claim71, wherein the CM comprises the amino acid sequence LSGRSDNH (SEQ IDNO: 13).
 82. The method of claim 71, wherein the activatable antibodycomprises a first linking peptide (LP1) and a second linking peptide(LP2), and wherein the activatable antibody in an uncleaved state hasthe structural arrangement from N-terminus to C-terminus as follows:MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
 83. The method of claim 82,wherein at least one of LP1 or LP2 comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GGS)_(n), (GSGGS)_(n)(SEQ ID NO: 15) and (GGGS)_(n) (SEQ ID NO: 16), where n is an integer ofat least one.
 84. The method of claim 82, wherein at least one of LP1 orLP2 comprises an amino acid sequence selected from the group consistingof GGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGSG (SEQ ID NO: 19),GSGGG (SEQ ID NO: 20), GGGSG (SEQ ID NO: 21), GSSSG (SEQ ID NO: 22),GSSGGSGGSGGSG (SEQ ID NO: 23), GSSGT (SEQ ID NO: 24), and GSSG (SEQ IDNO: 37).
 85. The method of claim 71, wherein the activatable antibody inan uncleaved state comprises a spacer, wherein the spacer is joineddirectly to the MM and has the structural arrangement from N-terminus toC-terminus of spacer-MM-CM-AB.
 86. The method of claim 85, wherein thespacer comprises the amino acid sequence QGQSGQ (SEQ ID NO: 38).
 87. Themethod of claim 73, wherein the agent is conjugated to the AB via alinker.
 88. The method of claim 87, wherein the linker is a cleavablelinker.
 89. The method of claim 82, wherein LP1 comprises the amino acidsequence GSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acidsequence GSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37).
 90. The methodof claim 71, wherein the activatable antibody comprises a first linkingpeptide (LP1) and a second linking peptide (LP2) such that theactivatable antibody in an uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-ABor AB-LP2-CM-LP1-MM, wherein LP1 comprises the amino acid sequenceGSSGGSGGSGGSG (SEQ ID NO: 23) and LP2 comprises the amino acid sequenceGSSGT (SEQ ID NO: 24) or GSSG (SEQ ID NO: 37), and wherein the ABcomprises a heavy chain amino acid sequence comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO:30, and SEQ ID NO: 34, and a light chain amino acid sequence comprisingthe amino acid sequence of SEQ ID NO: 68.